Combination therapy with MDM2 and EFGR inhibitors

ABSTRACT

Provided is a method of treating a proliferative disease, condition, or disorder in a subject by administering a combination of an inhibitor of p53 and MDM2 binding and an EGFR inhibitor. Various embodiments of the disclosed methods provide a synergistic anti-proliferative or anti-apoptotic effect compared to administration of one agent alone.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Divisional of U.S. Nonprovisionalapplication Ser. No. 13/188,351 filed 21 Jul. 2011, which is aContinuation in Part of U.S. Nonprovisional application Ser. No.12/986,146 filed 6 Jan. 2011, now U.S. Pat. No. 8,618,302 issued 31 Dec.2013, which claims the benefit of U.S. Provisional Application Ser. No.61/292,776 filed 6 Jan. 2010; the present application also is aContinuation in Part of International Application No. PCT/US11/20414filed 6 Jan. 2011 and International Application No. PCT/US11/20418 filed6 Jan. 2011; the present application also claims the benefit of U.S.Provisional Application Ser. No. 61/366,480 filed 21 Jul. 2010, all ofwhich are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention generally relates to development of new chemicalentities for use in the treatment of disease, and more particularly tomethods of identifying lead molecules for use in quasi-rational drugdesign.

BACKGROUND OF THE INVENTION

Tumor protein 53 (P53) is a tumor suppressing protein that regulates thecell cycle, suppresses tumors, and thereby prevents cancer. Murinedouble minute 2 (MDM2) is an important negative regulator of p53 andinhibitor of p53 transcriptional activation (see Vassilev 2006 Trends inMolecular Medicine 13(1), 23-31). MDM2 binds and inactivates p53 bydirectly blocking the p53 transactication domain and by serving as an E3ubiquitin ligase for p53, thereby targeting p53 protein forubiquitin-dependent degradation in the proteasome. About 11 millioncancer patients have an inactivating mutation in the p53 protein.

EGFR is involved in the same cellular signaling pathway as MDM2. EGFR isa known cancer-associated molecule and EGFR inhibitors, such as Tarceva,provide targeted cancer treatment. Significant numbers of cancerpatients become resistant to treatment with approved EGFR inhibitors,such as Tarceva. There is currently no approach to overcome suchresistance.

SUMMARY OF THE INVENTION

Disclosed herein are small molecule MDM2 inhibitor compounds useful forcancer treatment alone or in synergistic combination with an inhibitorof Epidermal Growth Factor Receptor (EGFR).

An MDM2 inhibitor used in combination with an EGFR inhibitor, such asTarceva, can provide treatment for patients with developed resistance.Combinatorial treatment with an MDM2 inhibitor and an EGFR inhibitor,such as Tarceva, can have synergistic anti-cancer effects and canovercome developed resistance.

One aspect provides a method of treating a proliferative disease,disorder, or condition. The method of combinatorial treatment caninclude administering to an MDM2 inhibitor and an EGFR inhibitor to asubject. The subject can be in need of such treatment. The amount of theMDM2 inhibitor and the EGFR inhibitor can be an amount sufficient toproduce a therapeutic effect.

In some embodiments, the proliferative disease, disorder, or conditionincludes cancer. In some embodiments, administering the MDM2 inhibitorand the EGFR inhibitor results in a synergistic reduction in cellproliferation in a tumor of the subject or a synergistic increase inapoptosis in a tumor of the subject as compared to administration ofeither the MDM2 inhibitor or the EGFR inhibitor alone.

In some embodiments, a pharmaceutical composition comprising an MDM2inhibitor, an EGFR inhibitor, and a pharmaceutically acceptable carrieror excipient is administered to the subject. In some embodiments, afirst pharmaceutical composition comprising an MDM2 inhibitor and apharmaceutically acceptable carrier or excipient and a secondpharmaceutical composition comprising an EGFR inhibitor and apharmaceutically acceptable carrier or excipient is administered to thesubject.

In some embodiments, the subject has one or more of (i) an inactivatingP53 mutation or deletion in the subject; (ii) a defect in an upstreamcomponent of a p53 pathway; (iii) a defect in a downstream component ofthe p53 pathway; (iv) increased expression an MDM2 gene as compared to acontrol; (v) increased levels of MDM2 protein as compared to a control;or (vi) resistance to treatment with an EGFR inhibitor alone. In someembodiments, the method includes selecting or modifying a treatment onthe basis of detecting in a subject one or more of (i) an inactivatingP53 mutation or deletion in the subject; (ii) a defect in an upstreamcomponent of a p53 pathway; (iii) a defect in a downstream component ofthe p53 pathway; (iv) increased expression an MDM2 gene as compared to acontrol; (v) increased levels of MDM2 protein as compared to a control;or (vi) resistance to treatment with an EGFR inhibitor alone.

In some embodiments, the EGFR inhibitor is selected from the groupconsisting of cetuximab, panitumumab, nimotuzumab, zalutumumab,matuzumab, potato carboxypeptidase inhibitor, gefitinib, lapatinib, anderlotinib, or a combination thereof. In some embodiments, the EGFRinhibitor is erlotinib (tradename Tarceva).

In some embodiments, the MDM2 inhibitor (i) inhibits MDM2 activity; (ii)increases phosphorylated p53; (iii) re-activates p53; (iv) inhibitsbinding of p53 and MDM2; or a combination thereof. In some embodiments,the MDM2 inhibitor inhibits binding of p53 and MDM2.

In some embodiments, the MDM2 inhibitor comprises a compound of Formula(2) as defined herein. In some embodiments, the MDM2 inhibitor comprisesa compound of Formula (10) as defined herein. In some embodiments, theMDM2 inhibitor comprises a compound of Formula (11) as defined herein.

Another aspect provides a pharmaceutical composition including an MDM2inhibitor; an EGFR inhibitor, and a pharmaceutically acceptable carrieror excipient.

Other objects and features will be in part apparent and in part pointedout hereinafter.

DESCRIPTION OF THE DRAWINGS

Those of skill in the art will understand that the drawings, describedbelow, are for illustrative purposes only. The drawings are not intendedto limit the scope of the present teachings in any way.

FIG. 1 is a line and scatter plot showing inhibition of p53/MDM2binding. RLU is shown as a function of nm, with EC₅₀ determined for eachof AD4-1505, AD4-10963, AD4-11511, AD4-10482, AD4-10942, AD4-10944, andAD4-10628.

FIG. 2 is a series of bar graphs showing percent inhibition ofproliferation in A431 cells resulting from a combination of Tarceva andeach of compounds AD4-10483, AD4-1505, AD4-10963, and AD4-10628-2. CI,an indication of synergy when less than 0.8, was determined for eachcombination.

FIG. 3 is a line and scatter plot showing CI of Tarceva as a function ofinhibition of p53/MDM2 binding (IC50), which demonstrates a correlationthereof.

FIG. 4 is a bar graph showing the anti-proliferative effect in A431cells of a Tarceva, AD4-10483, and a combination thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is based, at least in part, on the discovery thatcombinatorial administration of a compound that inhibits binding of p53and MDM2 along with an EGFR inhibitor (e.g., Tarceva) can providesynergistic anti-cancer effects and can overcomes developed resistance.A compounds ability to inhibit binding of p53 and MDM2 can be anindicator of synergistic anti-cancer effect when in combination with anEGFR inhibitor, especially in a subject with or at risk for resistanceto such EGFR inhibitor.

U.S. application Ser. No. 11/626,324, published as US ApplicationPublication No. 2008/0015194, is hereby incorporated by reference in itsentirety. U.S. application Ser. No. 12/986,146 is hereby incorporated byreference in its entirety. PCT/US2011/020414, published as WO2011/085126, is hereby incorporated by reference in its entirety.PCT/US2011/020418, published as WO 2011/085129, is hereby incorporatedby reference in its entirety.

A second target in cancer treatment is EGFR, a protein frequentlydysregulated in cancer cells. Overexpression of EGFR is known to beassociated with cancers of many tissues. Approved EGFR inhibitors, suchas Tarceva, provide anti-cancer effects but patients can developresistance.

oEGFR stimulates several cellular pathways, including the ras/rafpathway which leads to the subsequent stimulation of MEK and ERKinteraction with p53-MDM2 complex.

Further, as described herein, combinatorial administration of a compoundthat inhibits p53 binding to MDM2 and an EGFR inhibitor (e.g., Tarceva)can provide synergistic anti-cancer effects and can overcome developedresistance. Further, administration of MDM2 inhibitors in combinationwith an EGFR inhibitor, such as Tarceva, can result in synergistic tumorreduction and can overcome resistance to the EGFR inhibitor. The EGFRand p53/MDM2 signaling are interconnected through the interaction of ERKand MDM2. Provided herein are methods and materials to identify novelsmall molecule drug candidates that inhibit MDM2-p53 binding anddemonstrate synergystic anti-cancer effects with EGFR kinase inhibitors(e.g., approved EGFR kinase inhibitors).

Working examples provided herein show at least the following. A compoundwith the ability to inhibit p53/MDM2 binding can: (a) inhibit cellproliferation in the A431 cell line that over-expresses the EGFreceptor; (b) produce a synergistic effect with Tarceva, an EGFR kinaseinhibitor, in a cell proliferation assay in the A431 cell line; (c)induce apoptosis in the A431 cell line as measured by an increase incaspase activity, to a similar extent as Tarceva; (d) produce asynergistic effect with Tarceva in an apoptosis assay in the A431 cellline; (e) increases apoptosis in the A549 cell line, as measured byincreased DNA fragmentation; (f) produce a synergistic effect withTarceva in an apoptosis assay in the A431 cell line; or (g) increasephosphorylated p53; or a combination thereof. Examples 1-7 describeassays used in further examples. Examples 8-9 show compounds inhibitEFG-mediated cell proliferation and have a synergistic effect withTarceva. Examples 10-13 show compounds inhibit interaction of p53 andMDM2. Example 14 shows correlation of p53 inhibition and synergisticcell proliferation. Examples 15-18 show compounds induce apoptosis (asmeasured by DNA fragmentation assay and caspase assay). Example 17 showscompounds increase phosphorylated p53 compounds.

Inhibitor of P53/MDM2 Binding

The present disclosure provides several novel classes of compounds thatinhibit MDM2, re-activate p53, or inhibit the binding of p53 and MDM2. Apharmacophore-based class of compounds that inhibits MDM2 and therebyre-activates p53 has been identified. Such compounds can be used aloneas anti-cancer therapeutic agents, or in synergistic combination withtherapeutic EGFR inhibitors, as described further herein. Also providedherein are in silico pharmacophore-based design, in vitro assays, and invivo animal models to identify and optimize compounds that inhibit MDM2.

MDM2 is an inhibitor of activation of P53, which is a tumor suppressingprotein that prevents cancer. Over-expression of MDM2 can causeinactivation of tumor suppressor p53, which can result in many types ofcancer. A drug inhibiting MDM2 (e.g., inhibition of MDM2-p53 binding)can re-activate or restore the function of p53, resulting in tumorreduction and providing therapeutic approach for cancer treatment.

An MDM2 inhibitor, as that term is used herein, can refer to anyone of,or a combination of, MDM2 activity inhibition, increase ofphosphorylated p53, re-activation of p53, or inhibition of the bindingof p53 and MDM2. An MDM2 inhibitor can inhibit the activity of MDM2. AnMDM2 inhibitor can increase of phosphorylated p53. An MDM2 inhibitor canre-activate p53. An MDM2 inhibitor can inhibit p53/MDM2 binding.Increase of a phosphorylated form of p53 can decrease binding of p53 andMDM2.

An MDM2 inhibitor compound can be identified through one or more of thefollowing: (a) ability to inhibit cell proliferation in the A431 cellline that over-expresses the EGF receptor; (b) ability to produce asynergistic effect with Tarceva, an EGFR kinase inhibitor, in a cellproliferation assay in the A431 cell line; (c) ability to induceapoptosis in the A431 cell line as measured by an increase in caspaseactivity; (d) ability to produce a synergistic effect with Tarceva in anapoptosis assay in the A431 cell line; (e) ability to increasesapoptosis in the A549 cell line, as measured by increased DNAfragmentation; (f) ability to induce apoptosis in the A431 cell line toa similar extent as Tarceva; and (g) ability to increase phosphorylatedp53. Guidance as to pertinent assays for demonstration of the above areprovided in Examples 1-7. Exemplary results using such assays to assesscandidate compounds are provided in: Examples 8-9 (inhibition ofEFG-mediated cell proliferation and synergy with EGFR inhibitor);Examples 10-13 (inhibition of interaction of p53 and MDM2); Example 14(correlation of p53 inhibition and synergistic cell proliferation);Examples 15-18 (induction of apoptosis as measured by DNA fragmentationassay and caspase assay); and Example 17 (increased phosphorylated p53).

One of ordinary skill will understand that any compound demonstratingactivity as described above can be used in the combinatorial therapeuticapproach described herein. Exemplary MDM2 inhibitor compounds arefurther discussed below.

An MDM2 inhibitor can be a compound as disclosed in U.S. applicationSer. No. 11/626,324, published as US Application Publication No.2008/0015194; U.S. Nonprovisional application Ser. No. 12/986,146;International Application No. PCT/US11/20414, published as WO2011/085126; or International Application No. PCT/US11/20418, publishedas WO 2011/085129; each of which is incorporated herein by reference.

An MDM2 inhibitor can be a compound as disclosed in Vassilev 2006 Trendsin Molecular Medicine 13(1), 23-31. For example, an MDM2 inhibitor canbe a nutlin (e.g., a cis-imidazole compound, such as nutlin-3a); abenzodiazepine as disclosed in Grasberger et al. 2005 J Med Chem 48,909-912; a RITA compound as disclosed in Issaeva et al. 2004 Nat Med 10,1321-1328; a spiro-oxindole compound as disclosed in Ding et al. 2005 JAm Chem Soc 127, 10130-10131 and Ding et al. 2006 J Med Chem 49,3432-3435; or a quininol compound as disclosed in Lu et al. 2006 J MedChem 49, 3759-3762. As a further example, an MDM2 inhibitor can be acompound as disclosed in Chene 2003 Nat. Rev. Cancer 3, 102-109; Fotouhiand Graves 2005 Curr Top Med Chem 5, 159-165; or Vassilev 2005 J MedChem 48, 4491-4499.

Type A AD4-1505-like compounds.

An MDM2 inhibitor can be a compound according to Formula 2 (a Type AAD4-1505-like compound) as follows:

In the above structure, X¹ of Formula (2) can represent one or morefunctional group from the following Hydrogen atom, 2-Methyl, 5-Chloro,5-Nitro, or 6-Hydroxyl group.

R¹ of Formula (2) can represent:

a 2-Pyridyl ring of Formula (3) wherein R²³ is selected from the groupconsisting of hydrogen; fluoro; chloro; trifluoromethyl; methyl; ethyl;and methoxy; R³ is selected from the group consisting of hydrogen;fluoro; chloro; methyl; ethyl; methoxy; a straight chain or branched C-1to C-4 lower alkyl optionally containing unsaturation; a C-1 to C-6cycloalkyl optionally containing unsaturation or one oxygen or nitrogenatom; aryl comprising a phenyl or heteroaryl five or six membered ringcontaining from 1 to 4 N, O, or S atoms; and alkoxy —OR¹⁰ where R¹⁰ is astraight chain or branched C-1 to C-4 lower alkyl optionally containingunsaturation or a C-1 to C-6 cycloalkyl optionally containingunsaturation or one oxygen or nitrogen atom; R²⁴ is selected from thegroup consisting of: hydrogen; fluoro; chloro; and trifluoromethyl; andR⁴ is selected from the group consisting of hydrogen; methyl; a straightchain or branched C-1 to C-4 lower alkyl optionally containingunsaturation; a C-1 to C-6 cycloalkyl optionally containing unsaturationor one oxygen or nitrogen atom; aryl comprising a phenyl or heteroarylfive or six membered ring containing from 1 to 4 N, O, or S atoms; andalkoxy —OR¹⁰ where R¹⁰ is a straight chain or branched C-1 to C-4 loweralkyl optionally containing unsaturation or a C-1 to C-6 cycloalkyloptionally containing unsaturation or one oxygen or nitrogen atom;

a 3-Pyridyl ring of Formula (4) wherein R⁵, R⁶, and R⁷ are independentlyselected from the group consisting of: lower alkyl defined as C-1 toC-4, straight chain, branched, or optionally containing unsaturation,cycloalkyl defined as C-1 to C-6 optionally containing unsaturation,Aryl including phenyl or heteroaryl containing from 1 to 4 N, O, or Satoms, Alkoxy (—OR¹⁰ where R¹⁰ is defined as a lower alkyl group orcycloalkyl group in the above definition) (e.g., AD4-12908, AD4-13051,AD4-13021, AD4-13021, AD4-13063, AD4-013064, AD4-13065, AD4-13066,AD4-13101);

a 4-Pyridyl ring of Formula (5) wherein R⁸ and R⁹ are independentlyselected from the group consisting of: lower alkyl defined as C-1 toC-4, straight chain, branched, or optionally containing unsaturation,cycloalkyl defined as C-1 to C-6 optionally containing unsaturation,Aryl including phenyl or heteroaryl containing from 1 to 4 N, O, or Satoms, Alkoxy (—OR¹⁰ where R¹⁰ is defined as a lower alkyl group orcycloalkyl group in the above definition);

an unsubstituted phenyl ring or, preferably, a phenyl ring substitutedwith one or more of the following groups: lower alkyl defined as C-1 toC-4, straight chain, branched, or optionally containing unsaturation,cycloalkyl defined as C-1 to C-6 optionally containing unsaturation,Aryl including phenyl or heteroaryl containing from 1 to 4 N, O, or Satoms, alkoxy (—OR¹⁰ where R¹⁰ is defined as a lower alkyl group orcycloalkyl group as in the above definition), trifluoromethyl,trifluoromethoxy, difluoromethoxy, 3,4-methylenedioxy,2,3-methylenedioxy, Nitro or Halogen (F, Cl, Br, I); or

an unsubstituted heteroaryl five or six membered ring containing from 1to 4 N, O, or S atoms, or a heteroaryl five or six membered ringcontaining from 1 to 4 N, O, or S atoms which has one or more optionalsubstitution with the substituent defined as one or more of thefollowing groups: lower alkyl defined as C-1 to C-4, straight chain,branched, or optionally containing unsaturation, cycloalkyl defined asC-1 to C-6 optionally containing unsaturation, Aryl including phenyl orheteroaryl five or six membered ring containing from 1 to 4 N, O, or Satoms, Alkoxy (—OR¹⁰ where R¹⁰ is defined as a lower alkyl group orcycloalkyl group in the above definition).

It has been found that where R¹ is a 2-pyridyl ring of Formula (3) andR²⁴ is chloro or R²³ is methyl, the resulting compound can exhibitincreased stability.

It has been found that where R¹ is a 2-pyridyl ring of Formula (3)having combinations of substituted halogens and alkyl groups, theresulting compound can exhibit increased antiproliferative activity. Forexample, where R¹ is a 2-pyridyl ring of Formula (3), the followingsubstitutions can provide increased antiproliferative activity: R⁴ ishydrogen, R²⁴ is fluoro, R³ is hydrogen, and R²³ is fluoro; R⁴ ismethyl, R²⁴ is chloro, R³ is hydrogen, and R²³ is fluoro; R⁴ ishydrogen, R²⁴ is chloro, R³ is ethyl, and R²³ is fluoro; R⁴ is hydrogen,R²⁴ is fluoro, R³ is methyl, and R²³ is fluoro; R⁴ is hydrogen, R²⁴ ischloro, R³ is hydrogen, and R²³ is ethyl; R⁴ is methyl, R²⁴ is chloro,R³ is hydrogen, and R²³ is chloro; R⁴ is hydrogen, R²⁴ is chloro, R³ ismethyl, and R²³ is fluoro; R⁴ is hydrogen, R²⁴ is trifluoromethyl, R³ ishydrogen, and R²³ is hydrogen; R⁴ is hydrogen, R²⁴ is chloro, R³ ishydrogen, and R²³ is methyl; R⁴ is hydrogen, R²⁴ is chloro, R³ ishydrogen, and R²³ is chloro; R⁴ is hydrogen, R²⁴ is chloro, R³ ismethyl, and R²³ is hydrogen; or R⁴ is hydrogen, R²⁴ is chloro, R³ ischloro, and R²³ is hydrogen.

It has been found that where R¹ is a 2-pyridyl ring of Formula (3) andR²⁴ is chloro and there is additionally a chloro or methyl at one orboth of R³ or R²³, the resulting compound can exhibit increasedapoptosis. For example, where R¹ is a 2-pyridyl ring of Formula (3), thefollowing substitutions can provide increased apoptosis: R²⁴ is chloro,R³ is hydrogen, and R²³ is methyl; R²⁴ is chloro, R³ is methyl, and R²³is fluoro; R²⁴ is chloro, R³ is chloro, and R²³ is hydrogen; and R²⁴ ischloro, R³ is hydrogen, and R²³ is chloro.

It has been found that, where R¹ of Formula (2) is a 2-Pyridyl ring ofFormula (3), the group at R²⁴ of the aminopyridine can block metabolismin cultured hepatocytes.

As preferred examples, R¹ of Formula (2) can represent: an unsubstituted2-(1,3-thiazoyl) ring (see Formula (6)) or a 2-(1,3-thiazoyl) ring withgroups at the 4- or 5-position of the thiazole ring, for example a2-(4,5-Dimethyl-1,3-thiazoyl ring (see Formula (7)):

R² of Formula (2) can represent:

an unsubstituted Phenyl ring or a Phenyl ring substituted at the 2-, 3-,4-, 5- or 6-position with one or more of the following groups: loweralkyl defined as C-1 to C-4, straight chain, branched, or optionallycontaining unsaturation, cycloalkyl defined as C-1 to C-6 optionallycontaining unsaturation, Aryl including phenyl or heteroaryl containingfrom 1 to 4 N, O, or S atoms, Alkoxy (—OR¹⁰ where R¹⁰ is defined as alower alkyl group or cycloalkyl group as in the above definition),2,3-Methylenedioxy or 3,4-Methylenedioxy group, Dialkylamino (—NR₁₃R₁₄where R₁₃ and R₁₄ are independently selected from a Hydrogen atom orlower alkyl group as previously described); Trifluoromethyl,Trifluoromethoxy, Difluoromethoxy, 3,4-methylenedioxy,2,3-methylenedioxy, Nitro or Halogen (F, Cl, Br, I);

a 2-Thiophene ring of Formula (8) wherein R¹⁵, R¹⁶, and R¹⁷ areindependently selected from the group consisting of: hydrogen, loweralkyl, cycloalkyl, Alkoxy, Dialkylamino, Trifluoromethyl,Difluoromethyl, Trifluoromethoxy or halogen as described above;

a 3-Thiophene ring of Formula (9) wherein R¹⁸, R¹⁹, and R²⁰ areindependently selected from the group consisting of: lower alkyl,cycloalkyl, Alkoxy, Dialkylamino, Trifluoromethyl, Difluoromethyl,Trifluoromethoxy or halogen as described above;

an unsubstituted 2-Pyridyl ring or a 2-Pyridyl ring substituted at the4- or 6-position of the pyridine ring with one or more of the followinggroups: lower alkyl group as defined above, cycloalkyl group as definedabove;

an unsubstituted 3-Pyridyl ring or a 3-Pyridyl ring substituted at the2-, 4- or 6-position of the pyridine ring with one or more of thefollowing groups: lower alkyl group as defined above, cycloalkyl groupas defined above; or

an unsubstituted 4-Pyridyl ring or a 4-Pyridyl ring substituted at the2- or 6-position of the pyridine ring with one or more of the followinggroups: lower alkyl group as defined above, cycloalkyl group as definedabove.

It has been found that where R² is a phenyl ring substituted at the 2-and 4-positions, the resulting compound can exhibit increased stability.For example, where R² is 4-trifluoromethylphenyl;2-fluoro,4-trifluoromethylphenyl; or 2,4-dichlorophenyl, the resultingcompound can exhibit increased stability.

It has been found that where R² is a phenyl ring substituted with acombination of halogens and trifluoromethyl groups, the resultingcompound can exhibit increased antiproliferative activity. For example,where R² is 4-chlorophenyl; 2-fluoro,4-trifluoromethylphenyl;3-fluoro,4-chlorophenyl; 2-fluoro,4-chlorophenyl; 2,3-dichlorophenyl;2,3,5-trichlorophenyl; 2,4-dichlorophenyl; 3,4-dichlorophenyl; or3,5-dichlorophenyl, the resulting compound can exhibit increasedantiproliferative activity.

It has been found that where R² is a phenyl ring substituted at the 4position with chloro and additionally substituted at the 2- or3-position with chloro or fluoro, the resulting compound exhibitsincreased apoptosis. For example, where R² is 2,4-dichlorophenyl or2-chloro,4-fluorophenyl, the resulting compound can exhibits increasedapoptosis.

In some embodiments, the compound(s) are the enantiomeric isomers ofFormula (2).

In some embodiments, the compound(s) of Formula (2) are according to R¹and R² as provided in the following TABLE 1, TABLE 2, TABLE 3, and TABLE4.

TABLE 1 R1 and R2 substitution combinations (Pyr = pyridine; Ani =aniline) R2 phenyl R1 = 4Me— R1 = 4Me— substitution R1 = 5ClPyr R1 =5FPyr R1 = 4ClPyr 5ClPyr 5FPyr 2Cl AD4-13087 AD4-13104 AD4-13141AD4-13116 3Cl AD4-13151 4Cl AD4-13152 AD4-13157 2,3-diCl AD4-13086AD4-13103 AD4-13153 AD4-13126 3,4-diCl AD4-13054 AD4-13113 AD4-13069AD4-13166 AD4-13127 2,4-diCl AD4-13097 AD4-13110 AD4-13123 AD4-131282,5-diCl AD4-13095 AD4-13102 AD4-13158 AD4-13118 3,5-diCl AD4-13094AD4-13098 AD4-13122 AD4-13114 2,6-diCl AD4-13109 AD4-13120 AD4-13148AD4-13125 2,3,5-triCl AD4-13111 AD4-13132 AD4-13156 2Cl—4F AD4-13088AD4-13099 AD4-13149 AD4-13115 2Cl—6F AD4-13091 AD4-13112 AD4-13140AD4-13117 3F—4Cl 3Cl—4F 4CF3 AD4-13053 AD4-13044 AD4-13121 3F—4CF3AD4-13055 AD4-13061 AD4-13048 AD4-13106 2Cl—5CF3 AD4-13052 AD4-13049AD4-13060 4Cl—5CF3 AD4-13067 AD4-13071 AD4-13047 AD4-13108 2,4-diCF3AD4-13124 3CF3 AD4-13107 2F—4CF3 AD4-13046 AD4-13129 2,3,5,6-F4AD4-13070 AD4-13136 2,4-diF AD4-13050 AD4-13045 3-Me-4-OMe 2-F2,3,5,6-F4- 4-OCH2CF3 2-Me 3-F 4-OCF3 3-OH-4-OMe AD4-13186 2-OH-5-Me3,4-diOMe AD4-13194 2,3,4-triOMe AD4-13196

TABLE 2 R1 and R2 substitution combinations (Pyr = pyridine; Ani =aniline) R2 phenyl R1 = 3Me— R1 = 5- R1 = 4- R1 = 6- R1 = 3,5-substitution 5Cl CF3Pyr R1 = Pyr MePyr MePyr diFPyr 2Cl AD4-13134AD4-12907 AD4-12904 AD4-13183 3Cl AD4-13159 AD4-13173 4Cl AD4-13154AD4-13174 2,3-diCl AD4-13147 AD4-10051 AD4-12906 AD4-12905 3,4-diClAD4-13119 AD4-13030 AD4-13037 AD4-12917 AD4-12916 AD4-13182 2,4-diClAD4-13130 AD4-13033 AD4-13039 AD4-12912 AD4-12911 AD4-13175 2,5-diClAD4-13137 AD4-12910 AD4-12954 AD4-12955 AD4-13155 3,5-diCl AD4-13131AD4-12914 AD4-12915 AD4-12913 AD4-13176 2,6-diCl AD4-13142 AD4-13019AD4-13138 2,3,5-triCl AD4-13167 AD4-13072 AD4-13023 AD4-13181 2Cl—4FAD4-13139 AD4-13027 AD4-13026 AD4-13024 AD4-13146 2Cl—6F AD4-13135AD4-13020 AD4-12959 AD4-13133 3F—4Cl AD4-13229 3Cl—4F 4CF3 AD4-13041AD4-13028 AD4-10460 AD4-10486 AD4-10628 3F—4CF3 AD4-13043 AD4-13034AD4-13040 2Cl—5CF3 AD4-13058 AD4-13056 AD4-13035 4Cl—5CF3 AD4-13032AD4-13057 2,4-diCF3 3CF3 AD4-13164 AD4-12903 2F—4CF3 AD4-13042 AD4-13031AD4-13038 AD4-13096 2,3,5,6-F4 AD4-13059 AD4-12918 2,4-diF AD4-13068AD4-13029 AD4-13036 3-Me-4-OMe AD4-12965 2-F 2,3,5,6-F4- AD4-13093AD4-13092 AD4-13085 4-OCH2CF3 2-Me AD4-12935 3-F AD4-12953 4-OCF3AD4-12902 3-OH-4-OMe AD4-13190 AD4-1505 AD4-12909 2-OH-5-Me AD4-129363,4-diOMe AD4-13193 2,3,4-triOMe AD4-13208 2,4-diCl (2MeQ) AD4-13200

TABLE 3 R1 and R2 substitution combinations (Pyr = pyridine; Ani =aniline) R2 phenyl R1 = 3F— R1 = 5-Cl- R1 = 3-F-5- R1 = 4,5- R1 = 3-F-4-R1 = 3,5- substitution 5ClPyr 6-MePyr CF3Pyr diClPyr Me-5-ClPyrdiCl-6-MePyr 2Cl 3Cl AD4-13188 4Cl AD4-13161 AD4-13187 2,3-diClAD4-13172 AD4-13192 AD4-13211 3,4-diCl AD4-13150 AD4-13177 AD4-132022,4-diCl AD4-13143 AD4-13165 AD4-13178 AD4-13199 AD4-13206 2,5-diClAD4-13179 AD4-13220 3,5-diCl AD4-13189 AD4-13223 2,6-diCl 2,3,5-triClAD4-13209 AD4-13180 AD4-13213 2Cl—4F AD4-13185 2Cl—6F 3F—4Cl AD4-13224AD4-13230 3Cl—4F 4CF3 AD4-13162 3F—4CF3 AD4-13144 2Cl—5CF3 3CF3-4-ClAD4-13184 2,4-diCF3 3CF3 AD4-13145 2F—4CF3 2,3,5,6-F4 AD4-13163 2,4-diF3-Me-4-OMe 2-F 2,3,5,6-F4- 4-OCH2CF3 2-Me 3-F 4-OCF3 3-OH-4-OMeAD4-13191 AD4-13203 2-OH-5-Me 3,4-diOMe AD4-13195 2,3,4-triOMe AD4-13197AD4-13210

TABLE 4 R1 and R2 substitution combinations (Pyr = pyridine; Ani =aniline) R2 phenyl R1 = 2-Me- R1 = 3-Me- R1 = 3-MeO- R1 = 3-Et- R1 =3-F-4- R1 = 3,5- substitution 4-Cl-Ani 4-Cl-Ani 5-ClPyr 5-ClPyrEt-5-ClPyr diClPyr 2Cl 3Cl 4Cl AD4-13225 2,3-diCl AD4-13215 AD4-132223,4-diCl AD4-13204 AD4-13207 2,4-diCl AD4-13201 AD4-13217 AD4-13218AD4-13231 2,5-diCl AD4-13221 AD4-13227 3,5-diCl AD4-13216 AD4-132262,6-diCl 2,3,5-triCl AD4-13228 2Cl—4F AD4-13198 AD4-13205 2Cl—6F 3F—4Cl3Cl—4F 4CF3 3F—4CF3 2Cl—5CF3 3CF3-4-Cl 2,4-diCF3 3CF3 2F—4CF3 2,3,5,6-F42,4-diF 3-Me-4-OMe 2-F 2,3,5,6-F4- 4-OCH2CF3 2-Me 3-F 4-OCF3 3-OH-4-OMe2-OH-5-Me 3,4-diOMe 2,3,4-triOMe AD4-13214 AD4-13219

In some embodiments, the compound of Formula (2) is AD4-1505.

In some embodiments, the compound of Formula (2) is selected from acompound of TABLE 5.

TABLE 5 Exemplary Compounds of Formula (2)

AD4-12902

AD4-12903

AD4-12904

AD4-12905

AD4-12906

AD4-12907

AD4-12908

AD4-12909

AD4-12910

AD4-12911

AD4-12912

AD4-12913

AD4-12914

AD4-12915

AD4-12916

AD4-12917

AD4-12918

AD4-12935

AD4-12936

AD4-12937

AD4-12953

AD4-12954

AD4-12955

AD4-12958

AD4-12959

AD4-12965

AD4-12966

AD4-12990

AD4-12991

AD4-13018

AD4-13019

AD4-13020

AD4-13021A

AD4-13021B

AD4-13022

AD4-13023

AD4-13024

AD4-13025

AD4-13026

AD4-13027

AD4-13028

AD4-13029

AD4-13030

AD4-13031

AD4-13032

AD4-13033-1

AD4-13033-2

AD4-13034

AD4-13035

AD4-13036

AD4-13037

AD4-13038

AD4-13039

AD4-13040

AD4-13041

AD4-13042

AD4-13043

AD4-13044

AD4-13045

AD4-13046

AD4-13047

AD4-13048

AD4-13049

AD4-13050

AD4-13051

AD4-13052

AD4-13053

AD4-13054

AD4-13055

AD4-13056

AD4-13057

AD4-13058

AD4-13059

AD4-13060

AD4-13061

AD4-13062

AD4-13063

AD4-13064

AD4-13065

AD4-13066

AD4-13067

AD4-13068

AD4-13069

AD4-13070

AD4-13071

AD4-13072

AD4-13073

AD4-13074

AD4-13074-2

AD4-13075

AD4-13076

AD4-13077

AD4-13078

AD4-13079

AD4-13080

AD4-13081

AD4-13080

AD4-13081

AD4-13082

AD4-13083

AD4-13084

AD4-13085

AD4-13086

AD4-13087

AD4-13088

AD4-13089

AD4-13090

AD4-13091

AD4-13092

AD4-13093

AD4-13094

AD4-13095

AD4-13096

AD4-13097

AD4-13098

AD4-13099

AD4-13100

AD4-13101

AD4-13102

AD4-13103

AD4-13104

AD4-13105

AD4-13106

AD4-13107

AD4-13108

AD4-13109

AD4-13110

AD4-13111

AD4-13112

AD4-13113

AD4-13114

AD4-13115

AD4-13116

AD4-13117

AD4-13118

AD4-13119

AD4-13120

AD4-13121

AD4-13122

AD4-13123

AD4-13124

AD4-13125

AD4-13126

AD4-13127

AD4-13128

AD4-13129

AD4-13130

AD4-13131

AD4-13132

AD4-13133

AD4-13133

AD4-13134

AD4-13135

AD4-13136

AD4-13137

AD4-13138

AD4-13139

AD4-13140

AD4-13141

AD4-13142

AD4-13143

AD4-13144

AD4-13145

AD4-13146

AD4-13147

AD4-13148

AD4-13149

AD4-13150

AD4-13151

AD4-13152

AD4-13153

AD4-13154

AD4-13155

AD4-13156

AD4-13157

AD4-13158

AD4-13159

AD4-13160

AD4-13161

AD4-13162

AD4-13163

AD4-13164

AD4-13165

AD4-13166

AD4-13167

AD4-13172

AD4-13173

AD4-13174

AD4-13175

AD4-13176

AD4-13177

AD4-13178

AD4-13179

AD4-13180

AD4-13181

AD4-13182

AD4-13183

AD4-13184

AD4-13185

AD4-13186

AD4-13187

AD4-13188

AD4-13189

AD4-13190

AD4-13191

AD4-13192

AD4-13193

AD4-13194

AD4-13195

AD4-13196

AD4-13197

AD4-13198

AD4-13199

AD4-13200

AD4-13201

AD4-13202

AD4-13203

AD4-13204

AD4-13205

AD4-13206

AD4-13207

AD4-13208

AD4-13208

AD4-13209

AD4-13210

AD4-13211

AD4-13212

AD4-13213

AD4-13214

AD4-13215

AD4-13216

AD4-13217

AD4-13218

AD4-13219

AD4-13220

AD4-13221

AD4-13222

AD4-13223

AD4-13224

AD4-13225

AD4-13226

AD4-13227

AD4-13228

AD4-10484

AD4-10315

AD4-13229

AD4-13230

AD4-13231

AD4-10628

AD4-10963

In some embodiments, the compound(s) of Formula (2) excludes compoundAD4-1505, Formula (1).

In some embodiments, the compound(s) of Formula (2) excludes one or moreof the following compounds:

In some embodiments, for example methods of therapeutic treatment, thecompound(s) of Formula (2) can include one or more of the abovecompounds.

Type B AD4-1505-like compounds.

An MDM2 inhibitor can be a compound according to Formula 10 (a Type BAD4-1505-like compound) as follows:

In the above structure, X¹ and R² of Formula (10) are defined as abovefor structural sub-class Type A, Formula (2).

R²¹ of Formula (10) can represent:

a lower alkyl group with one to 6 carbons (C-1 to C-6), straight chain,branched, or optionally containing unsaturation, cycloalkyl defined asfive or six aliphatic ring (C-1 to C-6) optionally containingunsaturation;

an unsubstituted Phenyl ring or a Phenyl ring substituted at the 2-, 3-,4-, 5- or 6-position with one or more of the following groups: loweralkyl defined as C-1 to C-4, straight chain, branched, or optionallycontaining unsaturation, cycloalkyl defined as C-1 to C-6 optionallycontaining unsaturation, Aryl including phenyl or heteroaryl containingfrom 1 to 4 N, O, or S atoms, Alkoxy (—OR¹⁰ where R¹⁰ is defined as alower alkyl group or cycloalkyl group as in the above definition),2,3-Methylenedioxy or 3,4-Methylenedioxy group, Dialkylamino (—NR₁₃R₁₄,where R₁₃ and R₁₄ are independently selected from a Hydrogen atom orlower alkyl group as previously described); Trifluoromethyl,Trifluoromethoxy, Difluoromethoxy, 3,4-methylenedioxy,2,3-methylenedioxy, Nitro or Halogen (F, Cl, Br, I);

an unsubstituted 2-Pyridyl ring or a 2-Pyridyl ring substituted at the4- or 6-position of the pyridine ring with one or more of the followinggroups: lower alkyl group as defined above, cycloalkyl group as definedabove;

an unsubstituted 3-Pyridyl ring or a 3-Pyridyl ring substituted at the2-, 4- or 6-position of the pyridine ring with one or more of thefollowing groups: lower alkyl group as defined above, cycloalkyl groupas defined above;

an unsubstituted 4-Pyridyl ring or a 4-Pyridyl ring substituted at the2- or 6-position of the pyridine ring with one or more of the followinggroups: lower alkyl group as defined above, cycloalkyl group as definedabove; or

a heteroaryl five or six membered ring containing from 1 to 4 N, O, or Satoms.

In some embodiments, the compound(s) are the enantiomeric isomers ofFormula (10).

In some embodiments, the compound of Formula (10) is AD4-10950.

In some embodiments, the compound of Formula (10) is AD4-10960.

In some embodiments, the compound(s) of Formula (10) excludes compoundAD4-1505, Formula (1).

Type C AD4-1505-like

An MDM2 inhibitor can be a compound according to Formula 11 (a Type CAD4-1505-like compound) as follows:

In the above structure, X¹ and R² of Formula (11) are defined as abovefor structural sub-class Type A, Formula (2).

R²² of Formula (11) can represent a lower alkyl group with one to 6carbons (C-1 to C-6), straight chain, branched, optionally containingunsaturation, or substitution at the C-1 or C-2 carbons with one or moreof the following substituents: an unsubstituted Phenyl ring or a Phenylring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more ofthe following groups: lower alkyl defined as C-1 to C-4, straight chain,branched, or optionally containing unsaturation, cycloalkyl defined asC-1 to C-6 optionally containing unsaturation or one oxygen or nitrogenatom, Heteroaryl containing from 1 to 4 N, O, or S atoms, hydroxyl(—OH), Alkoxy (—OR¹⁰ where R¹⁰ is defined as a lower alkyl group orcycloalkyl group as in the above definition), Dialkylamino (—NR₁₃R₁₄,where R₁₃ and R₁₄ are independently selected from a Hydrogen atom orlower alkyl group as previously described); Trifluoromethyl,Trifluoromethoxy, Difluoromethoxy, or Halogen (F, Cl, Br, I).

A cycloalkyl is defined as five or six aliphatic ring (C-1 to C-6)optionally containing unsaturation or one oxygen or nitrogen atom.

In some embodiments, the compound(s) are the enantiomeric isomers ofFormula (11).

In some embodiments, the compound of Formula (11) is AD4-10535.

In some embodiments, the compound(s) of Formula (11) excludes compoundAD4-1505, Formula (1).

Structure and Function.

An MDM2 inhibitor compound described herein can have structural featuresassociated with one or more desired functions, such as stability,antiproliferative activity, or apoptotic activity.

It has been found that groups at the 5-position of the aminopyridine ofcompounds described herein provide analogs having increased stability(e.g., more stable toward liver microsome incubation). In someembodiments, a compound substituted at the 5-position of theaminopyridine can exhibit increased stability. For example, AD4-13053and AD4-13041 (both having a chlorine atom at the 5-position of theaminopyridine) show increased stability over AD4-10628. In someembodiments, a compound substituted with a chlorine atom at the5-position of the aminopyridine can exhibit increased stabilityincreases stability.

It has been found that combinations of halogens and alkyl groups on theaminopyridine ring of compounds described herein provide compounds withincreased antiproliferative activity. In some embodiments, a compoundwith the following aminopyridine ring substitutions provide increasedantiproliferative activity: 3,5-diF; 3-F,5-CL,6-Me; 3-F,5-C₁₋₆-Me;3-F,5-C₁₋₄-Et; and 3,5-diF,4-Me. In some embodiments, a compound withthe following aminopyridine ring substitutions provide further increasedantiproliferative activity: 3-Et,5-Cl; 3,5-diCl,6-Me; 3-F,5-C₁₋₄-Me; and5-CF₃. In some embodiments, a compound with the following aminopyridinering substitutions provide even further increased antiproliferativeactivity: 3-Me,5-Cl; 3,5-diCl; 4-Me,5-Cl; and 4,5-diCl.

It has been found that a chloro group at the 5-position of theaminopyridine ring and additional chloro or methyl groups at the 3- or4-positions on the aminopyridine ring of compounds described hereinprovide compounds with increased apoptotic activity. In someembodiments, a compound with the following aminopyridine ringsubstitutions provide increased apoptotic activity: 3-Me,5-Cl;3-F,5-C₁₋₄-Me; 4,5-diCl; and 3,5-diCl.

It has been found that groups at the 2- and 4-position of the benzenering of compounds described herein provide analogs having increasedstability (e.g., more stable toward liver microsome incubation). In someembodiments, a compound substituted at the 2- and 4-position of thebenzene ring of compounds can exhibit increased stability. For example,AD4-13041, AD4-13042, AD4-13165, and AD4-13206 show increased stability.In some embodiments, a compound substituted with a halogen atom at the2- or 4-position of the benzene ring of the aminopyridine can exhibitincreased stability increases stability. For example, a compoundsubstituted with a chlorine atom at the 2- and 4-position of the benzenering of the aminopyridine can exhibit increased stability increasesstability. As another example, a compound substituted with a fluorineatom at the 2- and 4-position of the benzene ring of the aminopyridinecan exhibit increased stability increases stability. As another example,a compound substituted with a trifluoromethyl at the 4-position or afluorine atom at the 2-position and a trifluoromethyl at the 4-positionof the benzene ring of the aminopyridine can exhibit increased stabilityincreases stability.

It has been found that combinations of halogens and trifluoromethylgroups on the benzene ring of compounds described herein providecompounds with increased antiproliferative activity. In someembodiments, a compound with the following benzene ring substitutionsprovide increased antiproliferative activity: 4-Cl; 2-F,4-CF₃; and3-F,4-Cl. In some embodiments, a compound with the following benzenering substitutions provide further increased antiproliferative activity:2-F,4-Cl; 2,3-diCl; and 2,3,5-triCl. In some embodiments, a compoundwith the following benzene ring substitutions provide even furtherincreased antiproliferative activity: 2,4-diCl; 3,4-diCl; and 3,5-diCl.

It has been found that a chloro group at the 4-position of the benzenering and additional chloro or fluoro groups at the 2- or 3-positions onthe benzene ring of compounds described herein provide compounds withincreased apoptotic activity. In some embodiments, a compound with thefollowing benzene ring substitutions provide increased apoptoticactivity: 2,4-diCl (see e.g., AD4-13130, AD4-13178); and 2-Cl, 4-F (seee.g., AD4-13185).

Synthesis.

Synthesis of the above described compounds can be as described in U.S.application Ser. No. 12/986,146 and WO 2011/085126. In brief, anAD4-1505-like compound can be synthesized by reacting an amino pyridineintermediate compound, an aldehyde intermediate compound and ahydroxyquinoline, as further described in U.S. application Ser. No.12/986,146 and WO 2011/085126. In some embodiments, the reaction caninclude combining the amino pyridine intermediate compound, the aldehydeintermediate compound and the hydroxyquinoline in ethanol (e.g.,absolute ethanol).

In some embodiments, the MDM2 inhibitor is selected from one or more ofthe following compounds:

Chemical Definitions

The expression “alkyl”, unless specifically limited, denotes a C₁₋₁₂alkyl group, suitably a C₁₋₆ alkyl group, e.g. C₁₋₄ alkyl group. Alkylgroups may be straight chain or branched. Suitable alkyl groups include,for example, methyl, ethyl, propyl (e.g. n-propyl and isopropyl), butyl(e.g. n-butyl, iso-butyl, sec-butyl and tert-butyl), pentyl (e.g.n-pentyl), hexyl (e.g. n-hexyl), heptyl (e.g. n-heptyl) and octyl (e.g.n-octyl). The expression “alk”, for example in the expressions “alkoxy”,“haloalkyl” and “thioalkyl” should be interpreted in accordance with thedefinition of “alkyl”. Exemplary alkoxy groups include methoxy, ethoxy,propoxy (e.g. n-propoxy), butoxy (e.g. n-butoxy), pentoxy (e.g.n-pentoxy), hexoxy (e.g. n-hexoxy), heptoxy (e.g. n-heptoxy) and octoxy(e.g. n-octoxy).

The expression “cycloalkyl”, unless specifically limited, denotes aC₃₋₁₀ cycloalkyl group (i.e., 3 to 10 ring carbon atoms), more suitablya C₃₋₈ cycloalkyl group, for example, a C₃₋₆ cycloalkyl group. Exemplarycycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl and cyclooctyl. A preferred number of ringcarbon atoms is three to six.

The expression “aryl”, unless specifically limited, denotes a C₆₋₁₂ arylgroup, suitably a C₆₋₁₀ aryl group, more suitably a C₆₋₈ aryl group.Aryl groups will contain at least one aromatic ring (e.g. one, two orthree rings). An example of a typical aryl group with one aromatic ringis phenyl. An example of a typical aryl group with two aromatic rings isnaphthyl.

The expression “heteroaryl”, unless specifically limited, denotes anaryl residue, wherein one or more (e.g., 1, 2, 3, or 4, suitably 1, 2 or3) ring atoms are replaced by heteroatoms selected from N, S and O, orelse a 5-membered aromatic ring containing one or more (e.g., 1, 2, 3,or 4, suitably 1, 2 or 3) ring atoms selected from N, S and O. Exemplarymonocyclic heteroaryl groups having one heteroatom include: fivemembered rings (e.g., pyrrole, furan, thiophene); and six membered rings(e.g., pyridine, such as pyridin-2-yl, pyridin-3-yl and pyridin-4-yl).Exemplary monocyclic heteroaryl groups having two heteroatoms include:five membered rings (e.g., pyrazole, oxazole, isoxazole, thiazole,isothiazole, imidazole, such as imidazol-1-yl, imidazol-2-ylimidazol-4-yl); six membered rings (e.g., pyridazine, pyrimidine,pyrazine). Exemplary monocyclic heteroaryl groups having threeheteroatoms include: 1,2,3-triazole and 1,2,4-triazole. Exemplarymonocyclic heteroaryl groups having four heteroatoms include tetrazole.Exemplary bicyclic heteroaryl groups include: indole (e.g., indol-6-yl),benzofuran, benzthiophene, quinoline, isoquinoline, indazole,benzimidazole, benzthiazole, quinazoline and purine.

A saturated group is generally understood as having no double or triplebonds. For example, in a saturated linear hydrocarbon, each carbon atomis attached to two hydrogen atoms, except those at the ends of thechain, which bear three hydrogen atoms. For example, an unsaturatedhydrocarbon is generally understood as a carbon structure containing oneor more double or triple bonds.

The term “halogen” or “halo” includes fluorine (F), chlorine (Cl)bromine (Br) or iodine (I).

The term “amino” refers to the group —NH₂.

All possible stereoisomers of the claimed compounds are included in thepresent disclosure. Where a compound described herein has at least onechiral center, it may accordingly exist as enantiomers. Where a compoundpossess two or more chiral centers it may additionally exist asdiastereomers. It is to be understood that all such isomers and mixturesthereof are encompassed within the scope of the present disclosure.

In view of the close relationship between the free compounds and thecompounds in the form of their salts, whenever a compound is referred toin this context, a corresponding salt is also intended, provided such ispossible or appropriate under the circumstances. The pharmaceuticallyacceptable salt can take a form in which a basic side chain isprotonated with an inorganic or organic acid. Representative organic orinorganic acids include hydrochloric, hydrobromic, perchloric, sulfuric,nitric, phosphoric, acetic, propionic, glycolic, lactic, succinic,maleic, fumaric, malic, tartaric, citric, benzoic, mandelic,methanesulfonic, hydroxyethanesulfonic, benzenesulfonic, oxalic, pamoic,2-naphthalenesulfonic, p-toluenesulfonic, cyclohexanesulfamic,salicylic, saccharinic or trifluoroacetic acid. Alternatively it maytake the form in which an acidic side chain forms a salt with a metalion (e.g., sodium, potassium ions and the like) or other positive ionsuch as ammonium. All pharmaceutically acceptable acid addition saltforms of the compounds described herein are intended to be embraced bythe scope of this disclosure.

Some of the crystalline forms of the compounds may exist in more thanone polymorphic form and as such all forms are intended to be includedin the present disclosure. In addition, some of the compounds may formsolvates with water (i.e., hydrates) or common organic solvents, andsuch solvates are also intended to be encompassed within the scope ofthis disclosure. The compounds, including their salts, can also beobtained in the form of their hydrates, or include other solvents usedfor their crystallization.

The present disclosure further includes within its scope prodrugs of thecompounds described herein. In general, such prodrugs will be functionalderivatives of the compounds which are readily convertible in vivo intothe desired therapeutically active compound. Thus, in these cases, themethods of treatment of the present invention, the term “administering”shall encompass the treatment of the various disorders described withprodrug versions of one or more of the claimed compounds, but whichconverts to the above specified compound in vivo after administration tothe subject.

As used herein, the term “composition” is intended to encompass aproduct comprising a claimed compound(s) in a therapeutically effectiveamount, as well as any product which results, directly or indirectly,from combinations of the claimed compounds.

EGFR Inhibitor

As shown herein, administration of an EGFR inhibitor in combination withan MDM2 inhibitors can result in synergistic inhibition proliferation orincreased death of cancer cells. An EGFR inhibitor can be ananti-proliferative or pro-apoptotic compound. An EGFR inhibitor can beselected so as to show a synergistic anti-proliferative or pro-apoptoticeffect when co-administered with an MDM2 inhibitor (e.g., an inhibitorof p53 and MDM2 binding). Quantitative methods (e.g., proteinphosphorylation detection) are known in the art to identify EGFRinhibitors and determine specificity and efficacy thereof (see e.g.,Olive 2004 Expert Rev Proteomics 1(3), 327-341).

An EGFR inhibitor can be a monoclonal antibody, such cetuximab(tradename Erbitux, IMC-C225); panitumumab (tradename Vectibix, INN,ABX-EGF); nimotuzumab (tradenames BIOMab EGFR, Theracim, Theraloc,CIMAher); zalutumumab (proposed tradename HuMax-EGFr); or matuzumab(formerly EMD 7200). Erbitux is a humanized monoclonal antibody thatbinds to an extracellular epitope on EGFR. Erbitux blocks activation ofthe receptor by preventing both ligand binding and receptordimerization. An EGFR inhibitor can be a protein, such as potatocarboxypeptidase inhibitor (PCI).

An EGFR inhibitor can be a small molecule inhibitor, such as gefitinib(tradename Iressa,N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine);lapatinib (tradename Tykerb,N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6-[5-[(2-ethylsulfonylethylamino)methyl]-2-furyl]quinazolin-4-amine);erlotinib (tradename Tarceva,N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine). Tykerb,Iressa, and Tarceva are kinase inhibitors that block EGFR tyrosinekinase activity.

In some embodiments, an EGFR inhibitor can be an selected from lapatinib(tradename Tykerb), gefitinib (tradename Iressa), erlotinib (tradenameTarceva), or cetuximab (tradename Erbitux), or a combination thereof.

Combination Therapy

As shown herein, administration of MDM2 inhibitors in combination withEGFR inhibitor Tarceva can result in synergistic inhibitionproliferation or increased death of cancer cells. Such a combinatorialtherapeutic approach can overcome resistance to conventional EGFRinhibitors, which have been reported to occur in a significant number ofpatients.

An MDM2 inhibitor optionally used in combination with an EGFR inhibitor,such as Tarceva, can provide treatment for patients with developedresistance. Such therapy can treat a variety of cancers, includingNSCLC, colon, pancreatic cancers and head and neck tumors, in patientswhere an EGFR inhibitor alone would not be effective.

Combinatorial treatment with an MDM2 inhibitor and an EGFR inhibitor canresult in a synergistic anti-cancer effect or can overcome developedresistance. Such combinatorial therapy is especially useful in a subjecthaving an inactivating P53 mutation, as described further herein.Synergistic effects or overcoming developed resistance can allow lowerdoses, significantly reducing therapy cost in a substantial patientpopulation.

Formulation

Embodiments of the compositions of the invention include pharmaceuticalformulations of the various compounds described herein.

The agents and compositions described herein can be formulated by anyconventional manner using one or more pharmaceutically acceptablecarriers or excipients as described in, for example, Remington'sPharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN:0781746736 (2005), incorporated herein by reference in its entirety.Such formulations will contain a therapeutically effective amount of abiologically active agent(s) described herein, preferably in purifiedform, together with a suitable amount of carrier so as to provide theform for proper administration to the subject.

The formulation should suit the mode of administration. The agents ofuse with the current disclosure can be formulated by known methods foradministration to a subject using several routes which include, but arenot limited to, parenteral, pulmonary, oral, topical, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, ophthalmic, buccal, and rectal. The individual agents may alsobe administered in combination with one or more additional agents ortogether with other biologically active or biologically inert agents.Such biologically active or inert agents may be in fluid or mechanicalcommunication with the agent(s) or attached to the agent(s) by ionic,covalent, Van der Waals, hydrophobic, hydrophilic or other physicalforces.

Controlled-release (or sustained-release) preparations may be formulatedto extend the activity of the agent(s) and reduce dosage frequency.Controlled-release preparations can also be used to effect the time ofonset of action or other characteristics, such as blood levels of theagent, and consequently affect the occurrence of side effects.Controlled-release preparations may be designed to initially release anamount of an agent(s) that produces the desired therapeutic effect, andgradually and continually release other amounts of the agent to maintainthe level of therapeutic effect over an extended period of time. Inorder to maintain a near-constant level of an agent in the body, theagent can be released from the dosage form at a rate that will replacethe amount of agent being metabolized or excreted from the body. Thecontrolled-release of an agent may be stimulated by various inducers,e.g., change in pH, change in temperature, enzymes, water, or otherphysiological conditions or molecules.

Agents or compositions described herein can also be used in combinationwith other therapeutic modalities, as described further below. Thus, inaddition to the therapies described herein, one may also provide to thesubject other therapies known to be efficacious for treatment of thedisease, disorder, or condition.

Therapeutic Methods

Another aspect provided herein is a process of treating a proliferativedisease, disorder, or condition with an MDM2 inhibitor and an EGFRinhibitor. Provided is a process of treating a proliferative disease,disorder, or condition in a subject in need administration of atherapeutically effective amount of an MDM2 inhibitor and an EGFRinhibitor, so as to increase apoptosis or decrease proliferation ofcancer cells, tumors, or tissue. In various embodiments, a proliferativedisease, disorder, or condition is associated with EGFR, MDM2, or p53.The therapeutic method can include administration of a therapeuticallyeffective amount of an MDM2 inhibitor and an EGFR inhibitor, either asindividual compositions or a jointly formulated composition.

An MDM2 inhibitor can be used with, or formulated with, knowntherapeutic compounds. Combination therapy is understood as atherapeutic regimen comprising, e.g., an MDM2 inhibitor described hereinand a second agent. an MDM2 inhibitor and a second agent can beformulated for separate administration or may be formulated foradministration together.

An MDM2 inhibitor can be combined with another anti-proliferativecompound, such as the EGFR kinase inhibitors, Tykerb, Iressa, andTarceva, or Erbitux, a humanized monoclonal antibody to the EGFreceptor, to produce a greater therapeutic effect than either agentalone. As shown herein, when MDM2 inhibitors were evaluated in a cellproliferation assay with EGFR inhibitors, the effect of the combinationof agents to inhibit cell proliferation was greater than the effect ofany of the agents alone. Specifically, compounds described herein wereevaluated with Tykerb, Iressa, Tarceva or Erbitux at a fixedconcentration ratio, which was ascertained from the results ofdose-response curves of each agent alone.

An MDM2 inhibitor can be used or formulated with an EGFR inhibitorapproved for treatment of an EGFR-related condition or disorder. Forexample, an MDM2 inhibitor can be used with or formulated with one ormore of Tykerb, Iressa, Tarceva, or Erbitux. Tykerb, Iressa, and Tarcevaare kinase inhibitors that block EGFR tyrosine kinase activity. Erbituxis a humanized monoclonal antibody that binds to an extracellularepitope on EGFR. Erbitux blocks activation of the receptor by preventingboth ligand binding and receptor dimerization. Thus, an MDM2 inhibitorand known EGFR inhibitors, such as those described above, can act in acomplementary or synergistic fashion.

Methods described herein are generally performed on a subject in needthereof. For example, a subject in need of the therapeutic methodsdescribed herein can be diagnosed with a proliferative disease,disorder, or condition, or at risk thereof. As another example, asubject in need of the therapeutic methods described herein can bediagnosed with a disease, disorder, or condition associated with EGFR,MDM2, or p53, or at risk thereof.

As another example, a subject in need of the therapeutic methodsdescribed herein can have, be diagnosed with, thought to have, orsuspected of having an inactivating mutation or deletion in p53 (e.g.,deletion or mutation of TP53 gene) (see Vassilev 2006 Trends inMolecular Medicine 13(1), 23-31). Determination of p53 status in tumorcells can be according to, for example, a DNA microarray-based p53GeneChip (see Ahrendt et al. 1999 Proc Natl Acad Sci USA 96, 7382-7387).

As another example, a subject in need of the therapeutic methodsdescribed herein can have, be diagnosed with, thought to have, orsuspected of having a defect in an upstream or a downstream component ofthe p53 pathway (see Vassilev 2006 Trends in Molecular Medicine 13(1),23-31).

As another example, a subject in need of the therapeutic methodsdescribed herein can have, be diagnosed with, thought to have, orsuspected of having aberrant MDM2 expression (see Vassilev 2006 Trendsin Molecular Medicine 13(1), 23-31). As a further example, a subject inneed of the therapeutic methods described herein can have, be diagnosedwith, thought to have, or suspected of having overexpression of the MDM2gene or overexpression of MDM2 protein without gene amplification, whichmay suppress p53 function (see Vassilev 2006 Trends in MolecularMedicine 13(1), 23-31).

As another example, a subject in need of the therapeutic methodsdescribed herein can have, be diagnosed with, thought to have, suspectedof having, or be at risk for a resistance to a conventional therapeutictreatment, such as treatment with an EGFR inhibitor.

A determination of the need for treatment can be assessed by a historyand physical exam consistent with the disease, disorder, or condition atissue. Diagnosis of the various conditions treatable by the methodsdescribed herein is within the skill of the art. The subject can be ananimal subject, preferably a mammal, more preferably horses, cows, dogs,cats, sheep, pigs, mice, rats, monkeys, guinea pigs, and chickens, andmost preferably a human.

Examples of proliferative diseases or conditions treatable withcompositions described herein include, but are not limited to, cancer;blood vessel proliferative disorders; fibrotic disorders; mesangial cellproliferative disorders; psoriasis; actinic keratoses; seborrheickeratoses; warts; keloid scars; eczema; and hyperproliferative diseasescaused by virus infections, such as papilloma virus infection.

Various compounds described herein can be effective for inhibiting EGFR,and thus, effective against diseases or conditions associated with EGFR,including, but not limited to, proliferative diseases. In someembodiments, the proliferative disease treated by a compound describedherein is a condition caused by excessive growth of cancer or non-cancercells that express a member of the EGFR family of receptors. The excesscells generated by a proliferative disease can express EGFR at normallevels or can overexpress EGFR. Particularly suitable diseases orconditions associated with EGFR can be those stimulated by a ligand ofEGFR or mutations of such ligands. Examples of such ligands thatstimulate EGFR include, but are not limited to, EGF, TGF-alpha,heparin-binding growth factor (HBGF), β-cellulin, and Cripto-1. Examplesof proliferative disease associated with EGFR include, but are notlimited to, cancer; blood vessel proliferative disorders; fibroticdisorders; mesangial cell proliferative disorders; psoriasis; actinickeratoses; seborrheic keratoses; warts; keloid scars; eczema; andhyperproliferative diseases caused by virus infections, such aspapilloma virus infection.

Cancer, or neoplasia, refers generally to any malignant neoplasm orspontaneous growth or proliferation of cells. A subject having “cancer”,for example, may have a leukemia, lymphoma, or other malignancy of bloodcells. In certain embodiments, the subject methods are used to treat asolid tumor. Exemplary solid tumors include but are not limited to nonsmall cell lung cancer (NSCLC), testicular cancer, lung cancer, ovariancancer, uterine cancer, cervical cancer, pancreatic cancer, colorectalcancer (CRC), breast cancer, as well as prostate, gastric, skin,stomach, esophageal, and bladder cancer.

Treatment of cancer or treating a subject having cancer can includeinhibition of replication of cancer cells, inhibition of spread ofcancer, reduction in tumor size, lessening or reducing the number ofcancerous cells in the body of a subject, or amelioration or alleviationof symptoms of cancer. A treatment can be considered therapeutic ifthere is a decrease in mortality or morbidity, and can be performedprophylactically, or therapeutically.

Methods described herein can be used to treat (e.g., reduce tumor size,decrease the vascularization, and/or increase the permeability of) anestablished tumor. An established tumor is generally understood as asolid tumor of sufficient size such that nutrients, e.g., oxygen, can nolonger permeate to the center of the tumor from the subject'svasculature by osmosis and therefore the tumor requires its own vascularsupply to receive nutrients. Methods described herein can be used totreat a solid tumor that is not quiescent and is actively undergoingexponential growth.

A therapeutic protocol can be modified according to permeability of asolid tumor. Permeability of a solid tumor generally refers to thepermeability of a solid tumor to a therapeutic. A solid tumor may besaid to be permeable to a therapeutic if the therapeutic is able toreach cells at the center of the tumor. An agent that increases thepermeability of a tumor may for example, normalize, e.g., maintain, thevasculature of a solid tumor. Tumor vascularization or tumorpermeability can be determined by a variety of methods known in the art,such as, e.g. by immunohistochemical analysis of biopsy specimens, or byimaging techniques, such as sonography of the tumor, computed tomography(CT) or magnetic resonance imaging (MRI) scans.

EGFR (Tuzi et al., 1991, Br. J. Cancer 63:227-233; Torp et al., 1992,APMIS 100:713-719) HER2/neu (Slamon et al., 1989, Science 244:707-712)and the PDGF-R (Kumabe et al., 1992, Oncogene 7:627-633) are known to beoverexpressed in many tumors and/or persistently activated by autocrineloops. Overexpression of the receptor and autocrine loops have beendemonstrated in most common and severe cancers (see e.g., Akbasak andSuner-Akbasak et al., 1992, J. Neurol. Sci. 111:119-133; Dickson et al.,1992, Cancer Treatment Res. 61:249-273; Korc et al., 1992, J. Clin.Invest. 90:1352-1360; Lee and Donoghue, 1992, J. Cell. Biol.118:1057-1070). Overexpression of EGFR is known to be associated withcancers of the bladder, brain, head and neck, pancreas, lung, breast,ovary, colon, prostate, and kidney. (see e.g., Atalay et al., 2003, Ann.Oncology 14:1346-1363; Herbst and Shin, 2002, Cancer 94:1593-1611;Modjtahedi et al., 1996, Br. J. Cancer 73:228-235). Overexpression ofEGFR can be correlated or associated with poor prognosis of the patients(see e.g., Herbst and Shin, 2002, Cancer 94:1593-1611; Modjtahedi etal., 1996, Br. J. Cancer 73:228-235). HER2 has been associated withbreast, ovarian, gastric, lung, pancreas and bladder cancer.

An inhibitor compound described herein can be used therapeuticallyeither as exogenous materials or as endogenous materials. Exogenousagents are those produced or manufactured outside of the body andadministered to the body. Endogenous agents are those produced ormanufactured inside the body by some type of device (biologic or other)for delivery to within or to other organs in the body.

According to the methods described herein, administration can beparenteral, pulmonary, oral, topical, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural,ophthalmic, buccal, or rectal administration.

When used in the treatments described herein, a therapeuticallyeffective amount of and MDM2 inhibitor and an EGFR inhibitor can beemployed in pure form or, where such forms exist, in pharmaceuticallyacceptable salt form and with or without a pharmaceutically acceptableexcipient. For example, the compounds of the present disclosure can beadministered, at a reasonable benefit/risk ratio applicable to anymedical treatment, in a sufficient amount to provide a sufficienttherapeutic outcome, as described further herein.

An effective amount of a compound described herein is generally thatwhich can exhibit an anti-proliferative effect to an extent such as toameliorate the treated condition. For example, an effective amount of acompound described herein may inhibit MDM2 or EGFR to an extent such asto ameliorate the treated condition. In some embodiments, an effectiveamount is that amount of therapy (or combination therapy) that issufficient to affect a desired result on a cancerous cell or tumor,including, but not limited to, for example, reducing tumor size,reducing tumor volume, decreasing vascularization of a solid tumor, orincreasing the permeability of a solid tumor to an agent, either invitro or in vivo. In certain embodiments, an effective amount of therapy(or combination therapy) is the amount that results in a percent tumorinhibition of more than about 55%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, orabout 100%. In certain embodiments, an effective amount of therapy (orcombination therapy) is sufficient to achieve a desired clinical result,including but not limited to, for example, ameliorating disease,stabilizing a subject, preventing or delaying the development of, orprogression of cancer in a subject. An effective amount of therapy (orcombination therapy) can be determined based on one administration orrepeated administration. Methods of detection and measurement of theindicators above are known to those of skill in the art. Such methodsinclude, but are not limited to measuring reduction in tumor burden,reduction of tumor size, reduction of tumor volume, reduction inproliferation of secondary tumors, decreased solid tumorvascularization, expression of genes in tumor tissue, presence ofbiomarkers, lymph node involvement, histologic grade, and nuclear grade.

In some embodiments, tumor burden can be determined. Tumor burden, alsoreferred to as tumor load, generally refers to a total amount of tumormaterial distributed throughout the body of a subject. Tumor burden canrefer to a total number of cancer cells or a total size of tumor(s),throughout the body, including lymph nodes and bone barrow. Tumor burdencan be determined by a variety of methods known in the art, such as, forexample, by measuring the dimensions of tumor(s) upon removal from thesubject, e.g., using calipers, or while in the body using imagingtechniques, e.g., ultrasound, computed tomography (CT) or magneticresonance imaging (MRI) scans. Tumor size can be determined, forexample, by determining tumor weight or tumor volume.

The amount of a composition described herein that can be combined with apharmaceutically acceptable carrier to produce a single dosage form willvary depending upon the host treated and the particular mode ofadministration. It will be appreciated by those skilled in the art thatthe unit content of agent contained in an individual dose of each dosageform need not in itself constitute a therapeutically effective amount,as the necessary therapeutically effective amount could be reached byadministration of a number of individual doses.

Toxicity and therapeutic efficacy of compositions described herein canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals for determining the LD₅₀ (the dose lethal to 50% ofthe population) and the ED₅₀, (the dose therapeutically effective in 50%of the population). The dose ratio between toxic and therapeutic effectsis the therapeutic index that can be expressed as the ratio LD₅₀/ED₅₀,where large therapeutic indices are preferred.

The specific therapeutically effective dose level for any particularsubject will depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration; the route of administration; the rate of excretion ofthe composition employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors well known in the medical arts (see e.g., Koda-Kimble etal. (2004) Applied Therapeutics: The Clinical Use of Drugs, LippincottWilliams & Wilkins, ISBN 0781748453; Winter (2003) Basic ClinicalPharmacokinetics, 4^(th) ed., Lippincott Williams & Wilkins, ISBN0781741475; Shargel (2004) Applied Biopharmaceutics & Pharmacokinetics,McGraw-Hill/Appleton & Lange, ISBN 0071375503). For example, it is wellwithin the skill of the art to start doses of agents at levels lowerthan those required to achieve the desired therapeutic effect and togradually increase the dosage until the desired effect is achieved. Ifdesired, the effective daily dose may be divided into multiple doses forpurposes of administration. Consequently, single dose compositions maycontain such amounts or submultiples thereof to make up the daily dose.It will be understood, however, that the total daily usage of thecompounds and compositions of the present disclosure will be decided byan attending physician within the scope of sound medical judgment.

Administration of an MDM2 inhibitor or an EGFR inhibitor as describedherein can occur as a single event, a periodic event, or over a timecourse of treatment. For example, agents can be administered daily,weekly, bi-weekly, or monthly. As another example, agents can beadministered in multiple treatment sessions, such as 2 weeks on, 2 weeksoff, and then repeated twice; or every 3rd day for 3 weeks. An MDM2inhibitor and an EGFR inhibitor can have the same or differentadministration protocols. One of ordinary skill will understand theseregimes to be exemplary and could design other suitable periodicregimes. For treatment of acute conditions, the time course of treatmentwill usually be at least several days. Certain conditions could extendtreatment from several days to several weeks. For example, treatmentcould extend over one week, two weeks, or three weeks. For more chronicconditions, treatment could extend from several weeks to several monthsor even a year or more.

Treatment in accord with the methods described herein can be performedprior to, concurrent with, or after conventional treatment modalitiesfor a disease, disorder, or condition associated with a targetbiomolecule for which the compound is specific.

A combination of an MDM2 inhibitor and an EGFR inhibitor can beadministered simultaneously or sequentially with another agent, such asan antibiotic, an antiinflammatory, or another agent. Simultaneousadministration can occur through administration of separatecompositions, each containing one or more of an MDM2 inhibitor, an EGFRinhibitor, an antibiotic, an antiinflammatory, or another agent.Simultaneous administration can occur through administration of onecomposition containing three or more of an MDM2 inhibitor, an EGFRinhibitor, an antibiotic, an antiinflammatory, or another agent. Acombination of an MDM2 inhibitor and an EGFR inhibitor can beadministered sequentially with an antibiotic, an antiinflammatory, oranother agent. For example, a combination of an MDM2 inhibitor and anEGFR inhibitor can be administered before or after administration of anantibiotic, an antiinflammatory, or another agent.

Administration

Agents and compositions described herein can be administered accordingto methods described herein in a variety of means known to the art. Theagents and composition can be used therapeutically either as exogenousmaterials or as endogenous materials. Exogenous agents are thoseproduced or manufactured outside of the body and administered to thebody. Endogenous agents are those produced or manufactured inside thebody by some type of device (biologic or other) for delivery within orto other organs in the body.

As discussed above, administration can be parenteral, pulmonary, oral,topical, intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectaladministration.

Agents and compositions described herein can be administered in avariety of methods well known in the arts. Administration can include,for example, methods involving oral ingestion, direct injection (e.g.,systemic or stereotactic), implantation of cells engineered to secretethe factor of interest, drug-releasing biomaterials, polymer matrices,gels, permeable membranes, osmotic systems, multilayer coatings,microparticles, implantable matrix devices, mini-osmotic pumps,implantable pumps, injectable gels and hydrogels, liposomes, micelles(e.g., up to 30 μm), nanospheres (e.g., less than 1 μm), microspheres(e.g., 1-100 μm), reservoir devices, a combination of any of the above,or other suitable delivery vehicles to provide the desired releaseprofile in varying proportions. Other methods of controlled-releasedelivery of agents or compositions will be known to the skilled artisanand are within the scope of the present disclosure.

Delivery systems may include, for example, an infusion pump which may beused to administer the agent or composition in a manner similar to thatused for delivering insulin or chemotherapy to specific organs ortumors. Typically, using such a system, an agent or composition isadministered in combination with a biodegradable, biocompatiblepolymeric implant that releases the agent over a controlled period oftime at a selected site. Examples of polymeric materials includepolyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid,polyethylene vinyl acetate, and copolymers and combinations thereof. Inaddition, a controlled release system can be placed in proximity of atherapeutic target, thus requiring only a fraction of a systemic dosage.

Agents can be encapsulated and administered in a variety of carrierdelivery systems. Examples of carrier delivery systems includemicrospheres, hydrogels, polymeric implants, smart polymeric carriers,and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006)Polymers in Drug Delivery, CRC, ISBN-10: 0849325331). Carrier-basedsystems for molecular or biomolecular agent delivery can: provide forintracellular delivery; tailor biomolecule/agent release rates; increasethe proportion of biomolecule that reaches its site of action; improvethe transport of the drug to its site of action; allow colocalizeddeposition with other agents or excipients; improve the stability of theagent in vivo; prolong the residence time of the agent at its site ofaction by reducing clearance; decrease the nonspecific delivery of theagent to nontarget tissues; decrease irritation caused by the agent;decrease toxicity due to high initial doses of the agent; alter theimmunogenicity of the agent; decrease dosage frequency, improve taste ofthe product; or improve shelf life of the product.

Screening

Also provided are methods for screening for an MDM2 inhibitor for use inthe combinatorial therapy described herein. Candidate substances forscreening according to the methods described herein include, but are notlimited to, fractions of tissues or cells, nucleic acids, polypeptides,siRNAs, antisense molecules, aptamers, ribozymes, triple helixcompounds, antibodies, and small (e.g., less than about 2000 mw, or lessthan about 1000 mw, or less than about 800 mw) organic molecules orinorganic molecules including but not limited to salts or metals.

Candidate molecules encompass numerous chemical classes, for example,organic molecules, such as small organic compounds having a molecularweight of more than 50 and less than about 2,500 Daltons. Candidatemolecules can comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group, andusually at least two of the functional chemical groups. The candidatemolecules can comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups.

A candidate molecule can be a compound in a library database ofcompounds. One of skill in the art will be generally familiar with, forexample, numerous databases for commercially available compounds forscreening (see e.g., ZINC database, UCSF, with 2.7 million compoundsover 12 distinct subsets of molecules; Irwin and Shoichet (2005) J ChemInf Model 45, 177-182). One of skill in the art will also be familiarwith a variety of search engines to identify commercial sources ordesirable compounds and classes of compounds for further testing (seee.g., ZINC database; eMolecules.com; and electronic libraries ofcommercial compounds provided by vendors, for example: ChemBridge,Princeton BioMolecular, Ambinter SARL, Enamine, ASDI, Life Chemicalsetc).

Candidate molecules for screening according to the methods describedherein include both lead-like compounds and drug-like compounds. Alead-like compound is generally understood to have a relatively smallerscaffold-like structure (e.g., molecular weight of about 150 to about350 kD) with relatively fewer features (e.g., less than about 3 hydrogendonors and/or less than about 6 hydrogen acceptors; hydrophobicitycharacter xlogP of about −2 to about 4) (see e.g., Angewante (1999)Chemie Int. ed. Engl. 24, 3943-3948). In contrast, a drug-like compoundis generally understood to have a relatively larger scaffold (e.g.,molecular weight of about 150 to about 500 kD) with relatively morenumerous features (e.g., less than about 10 hydrogen acceptors and/orless than about 8 rotatable bonds; hydrophobicity character xlogP ofless than about 5) (see e.g., Lipinski (2000) J. Pharm. Tox. Methods 44,235-249). Preferably, initial screening is performed with lead-likecompounds.

When designing a lead from spatial orientation data, it can be useful tounderstand that certain molecular structures are characterized as being“drug-like”. Such characterization can be based on a set of empiricallyrecognized qualities derived by comparing similarities across thebreadth of known drugs within the pharmacopoeia. While it is notrequired for drugs to meet all, or even any, of these characterizations,it is far more likely for a drug candidate to meet with clinicalsuccessful if it is drug-like.

Several of these “drug-like” characteristics have been summarized intothe four rules of Lipinski (generally known as the “rules of fives”because of the prevalence of the number 5 among them). While these rulesgenerally relate to oral absorption and are used to predictbioavailability of compound during lead optimization, they can serve aseffective guidelines for constructing a lead molecule during rationaldrug design efforts such as may be accomplished by using the methods ofthe present disclosure.

The four “rules of five” state that a candidate drug-like compoundshould have at least three of the following characteristics: (i) aweight less than 500 Daltons; (ii) a log of P less than 5; (iii) no morethan 5 hydrogen bond donors (expressed as the sum of OH and NH groups);and (iv) no more than 10 hydrogen bond acceptors (the sum of N and Oatoms). Also, drug-like molecules typically have a span (breadth) ofbetween about 8 Å to about 15 Å.

Kits

Also provided are kits. Such kits can include the compositions of thepresent invention and, in certain embodiments, instructions foradministration. Such kits can facilitate performance of the methodsdescribed herein, for example, treatment methodologies or screeningmethodologies. When supplied as a kit, the different components of thecomposition can be packaged in separate containers and admixedimmediately before use. Components include, but are not limited to oneor more compounds described herein, vectors, diagnostic reagents, assayreagents, and/or combinations thereof. Such packaging of the componentsseparately can, if desired, be presented in a pack or dispenser devicewhich may contain one or more unit dosage forms containing thecomposition. The pack may, for example, comprise metal or plastic foilsuch as a blister pack. Such packaging of the components separately canalso, in certain instances, permit long-term storage without losingactivity of the components.

Kits may also include reagents in separate containers such as, forexample, sterile water or saline to be added to a lyophilized activecomponent packaged separately. For example, sealed glass ampules maycontain a lyophilized component and in a separate ampule, sterile water,or sterile saline, each of which has been packaged under a neutralnon-reacting gas, such as nitrogen. Ampules may consist of any suitablematerial, such as glass, organic polymers, such as polycarbonate,polystyrene, ceramic, metal or any other material typically employed tohold reagents. Other examples of suitable containers include bottlesthat may be fabricated from similar substances as ampules, and envelopesthat may consist of foil-lined interiors, such as aluminum or an alloy.Other containers include test tubes, vials, flasks, bottles, syringes,and the like. Containers may have a sterile access port, such as abottle having a stopper that can be pierced by a hypodermic injectionneedle. Other containers may have two compartments that are separated bya readily removable membrane that upon removal permits the components tomix. Removable membranes may be glass, plastic, rubber, and the like.

In certain embodiments, kits can be supplied with instructionalmaterials. Instructions may be printed on paper or other substrate,and/or may be supplied as an electronic-readable medium, such as afloppy disc, mini-CD-ROM, CD-ROM, DVD-ROM, Zip disc, videotape, audiotape, and the like. Detailed instructions may not be physicallyassociated with the kit; instead, a user may be directed to an Internetweb site specified by the manufacturer or distributor of the kit.

Compositions and methods described herein utilizing molecular biologyprotocols can be according to a variety of standard techniques known tothe art (see, e.g., Sambrook and Russel (2006) Condensed Protocols fromMolecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols inMolecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929;Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3ded., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J.and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005)Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production ofRecombinant Proteins: Novel Microbial and Eukaryotic Expression Systems,Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein ExpressionTechnologies, Taylor & Francis, ISBN-10: 0954523253).

Definitions and methods described herein are provided to better definethe present disclosure and to guide those of ordinary skill in the artin the practice of the present disclosure. Unless otherwise noted, termsare to be understood according to conventional usage by those ofordinary skill in the relevant art.

In some embodiments, numbers expressing quantities of ingredients,properties such as molecular weight, reaction conditions, and so forth,used to describe and claim certain embodiments of the present disclosureare to be understood as being modified in some instances by the term“about.” In some embodiments, the term “about” is used to indicate thata value includes the standard deviation of the mean for the device ormethod being employed to determine the value. In some embodiments, thenumerical parameters set forth in the written description and attachedclaims are approximations that can vary depending upon the desiredproperties sought to be obtained by a particular embodiment. In someembodiments, the numerical parameters should be construed in light ofthe number of reported significant digits and by applying ordinaryrounding techniques. Notwithstanding that the numerical ranges andparameters setting forth the broad scope of some embodiments of thepresent disclosure are approximations, the numerical values set forth inthe specific examples are reported as precisely as practicable. Thenumerical values presented in some embodiments of the present disclosuremay contain certain errors necessarily resulting from the standarddeviation found in their respective testing measurements. The recitationof ranges of values herein is merely intended to serve as a shorthandmethod of referring individually to each separate value falling withinthe range. Unless otherwise indicated herein, each individual value isincorporated into the specification as if it were individually recitedherein.

In some embodiments, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment(especially in the context of certain of the following claims) can beconstrued to cover both the singular and the plural, unless specificallynoted otherwise. In some embodiments, the term “or” as used herein,including the claims, is used to mean “and/or” unless explicitlyindicated to refer to alternatives only or the alternatives are mutuallyexclusive.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and can also cover other unlisted steps. Similarly, anycomposition or device that “comprises,” “has” or “includes” one or morefeatures is not limited to possessing only those one or more featuresand can cover other unlisted features.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.“such as”) provided with respect to certain embodiments herein isintended merely to better illuminate the present disclosure and does notpose a limitation on the scope of the present disclosure otherwiseclaimed. No language in the specification should be construed asindicating any non-claimed element essential to the practice of thepresent disclosure.

Groupings of alternative elements or embodiments of the presentdisclosure disclosed herein are not to be construed as limitations. Eachgroup member can be referred to and claimed individually or in anycombination with other members of the group or other elements foundherein. One or more members of a group can be included in, or deletedfrom, a group for reasons of convenience or patentability. When any suchinclusion or deletion occurs, the specification is herein deemed tocontain the group as modified thus fulfilling the written description ofall Markush groups used in the appended claims.

Citation of a reference herein shall not be construed as an admissionthat such is prior art to the present disclosure.

Having described the present disclosure in detail, it will be apparentthat modifications, variations, and equivalent embodiments are possiblewithout departing the scope of the present disclosure defined in theappended claims. Furthermore, it should be appreciated that all examplesin the present disclosure are provided as non-limiting examples.

EXAMPLES

The following non-limiting examples are provided to further illustratethe present disclosure. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples that followrepresent approaches the inventors have found function well in thepractice of the present disclosure, and thus can be considered toconstitute examples of modes for its practice. However, those of skillin the art should, in light of the present disclosure, appreciate thatmany changes can be made in the specific embodiments that are disclosedand still obtain a like or similar result without departing from thespirit and scope of the present disclosure.

Example 1 Alphascreen Measurement of p53/MDM2 Interaction (UsingTruncated MDM2)

The following example describes an assay that measured the ability ofcompounds to inhibit the binding of p53 to MDM2 using the AlphaScreenassay technology (PerkinElmer). The following protocol is an adaptationof the method described by H. R. Lawrence et al. (Bioorg. Med. Chem.Lett. 19 (2009) 3756-3759). Recombinant, truncated, human, N-terminalGST-MDM2 (aa 1-150) was obtained from GeneScript. Wild-type, full lengthhuman N-terminal 6-his p53 was purchased from SignalChem.

Resulting in a final reaction volume of 24 μl PBS, 0.1% Tween-20, and10% glycerol, 30 ng of MDM2 was added, followed by the addition of 10 ofcompound diluted in 100% DMSO that provided a final DMSO concentrationof 4%. 30 ng of p53 was then added, mixed, and incubated at roomtemperature for 1 hour. Glutathione donor beads and Nickel acceptorbeads (0.5 μg each; PerkinElmer) were added under subdued lightingconditions to a final reaction volume of 30 μl/well in a 96 well, ½volume Proxima plate. The reaction was incubated at room temperaturewith shaking for 18 hrs in the dark. Analysis was then performed on aPerkinElmer EnSpire plate reader to determine the ability of compoundsto inhibit the binding of p53 to MDM2.

Example 2 DNA Fragmentation, Measurement of Apoptosis

The following example describes an assay that measured the ability ofcompounds to induce DNA fragmentation, an indicator of cell apoptosis,using the Roche Cell Death Detection ELISA kit (Cat #11920 685 001).Methods are according to Example 1 unless otherwise specified.

Tissue Culture.

On day 1, seed A549 cells (10,000 cells/well at 200 μl/well) in tissueculture media (RPMI-1640 with 1% sodium pyruvate, 1% Pen-Strep, 1%L-Glutamine and 10% FBS) were placed in 96-well, tissue culture-treatedplates. The plates were allowed to incubate overnight @ 37° C., 5% CO₂.On day 2, the media was removed from the plates and 160 μl mediacontaining 5% FBS was added. 40 μl of media-containing test compound in100% DMSO (prepared at 5× the dosing concentration) was added to theexisting media resulting in a final DMSO concentration of 0.5%. Cellswere incubated in the presence of a compound for 24 hrs @ 37° C., 5%CO₂.

DNA Fragmentation Assay.

After 24 hrs, the abovementioned plates were centrifuged at 200×g for 10min. The media was removed by gently inverting the plate and disposingof the media onto a paper towel. The plates were tapped gently to removeexcess media. 200 μl lysis buffer was added to each well, and mixed byshaking at 300 rpm. The plates were incubated at room temperature for 30min. The plates were centrifuged at 200×g for 10 min and 20 μl of lysissupernatant was removed for use in ELISA analysis to detect cell death.

ELISA Analysis.

20 μl of the cell lysis supernatant was placed into streptavidin-coatedplates with 20 μl positive control and 20 μl incubation buffer negativecontrol. Add 80 μl Immunoreagent (for DNA fragment detection) to eachwell. The wells were covered with foil adhesive and shaken at 300 rpmfor 2 h at room temperature. The supernatant solution was removed andthe wells were washed 3 times with 300 μl incubation buffer. 100 μl ofABTS detection substrate was added to each well. The wells wereincubated on a plate shaker at 250 rpm for approximately 10-20 min. 100μl ABTS of stop buffer was then added. The absorbance was read at 400and 492 nm on PolarStar plate reader. The absorbance measures thecolored product spectrophotometrically, correlating with apoptosis.

Example 3 Proteome Profile, Measurement of Apoptosis

This example describes an assay that measured the relative expressionlevels of 35 apoptosis-related proteins in a single sample of whole cellextract. Methods are according to Examples 1-2 unless otherwisespecified.

The protocol and reagents were purchased from R&D systems (Humanapoptosis array kit; cat. #ARY009). The kit consisted of an antibodyarray spotted on nitrocellulose membranes with each specific antibodyprinted in duplicate. The detection antibodies were biotinylated so theycould be used with a streptavidin-HRP conjugate designed forchemiluminescent imaging. The protocol was modified to utilize the LiCorinfrared imaging technology by substituting an infrared 680 nm-taggedstreptavidin.

Tissue Culture.

A549 NSCLC cells were seeded in 6 well tissue culture plates inRPMI-1640 supplemented with 5% FBS at a density of 1×10⁶ cells/well, 2ml/well and allowed to incubate overnight at 37° C., 5% CO₂, and 85%relative humidity.

The following day, two μl of compound dilutions in 100% DMSO were addedto the appropriate wells resulting in a final concentration of 5 μM. Theplates were then incubated for 6 hours at 37° C., 5% CO₂, and 85%relative humidity. Following incubation, the cells were washed with 2 mlof PBS, then lysed with 500 μl/well of Lysis buffer, supplemented with a1/200 dilution of mammalian protease inhibitor cocktail (Sigma Aldrich).The plates were shaken for 30 min at room temperature, spun @ 14,000×gfor 5 minutes. 250 μl of the supernatant was removed and added to 1.25ml of Assay buffer 1. The supernatant and Assay buffer 1 were incubatedwith membranes overnight at room temperature with shaking.

The following day, the membranes were washed 3 times for 10 min each,with 1× wash buffer in individual 50 ml Falcon tubes at room temperaturewith rotation. The membranes were incubated with a 1/100 dilution ofbiotinylated detection antibodies for 3 hours at room temperature withshaking.

The membranes were washed, then incubated with a 1/1000 dilution of IR680 nm-labeled streptavidin (LiCor) for 60 minutes with shaking in thedark. The membranes were again washed. Excess buffer was blotted frommembranes, placed between two pieces of transparent plastic and imagedon the LiCor infrared imager. The LiCor Odyssey software is used toquantitate the pixel intensity of each spot in the array.

Example 4 p53 In-Cell Western, Measurement of Phosphorylated p53

This example describes an assay that measures the relative expression ofhuman p53 and phospho-p53 (S15) in intact, formaldehyde-fixed, A549NSCLC cells in a 96 well format. Methods are according to Examples 1-3unless otherwise specified.

The procedure was adapted from a protocol suggested by LiCorBiosciences.

First, A549 NSCLC cells were seeded in black 96 well clear bottom tissueculture plates at a density of 25,000 cells/well, 200 μl/well, inRPMI-1640 supplemented with 5% FBS and allowed to incubate overnight at37° C., 5% CO₂, and 85% relative humidity.

One μl of compound titrations in DMSO was added to each well and allowedto incubate for 8 and 30 hrs at 37° C., 5% CO₂, and 85% relativehumidity for the determination of phospho p53(S15) and total p53,respectively. The media was removed, then 150 μl/well of 4% formaldehydein PBS was added and incubated at room temperature for 30 min.

The wells were washed 5× for 5 minutes each, using 200 μl/well of 0.1%Triton X-100 in PBS with shaking to ensure permeation of the cells. 150μl/well of Odyssey Blocking buffer (LiCor) was added to the wells andallowed to shake for 90 minutes at room temperature.

Next, the blocking buffer was removed, then incubated with 50 μl/well ofa 1/250 dilution of either: (1) polyclonal Goat anti-human p53 or; (2) a1/40 dilution of polyclonal Rbt anti human phospho (S15) p53 for 16 hrsat room temperature with shaking. The wells were washed 5× for 5 mineach with 0.1% Tween-20 in PBS with shaking.

The cells were then incubated with 50 μl/well of either: (1) a 1/5000dilution of Donkey anti Goat IgG IR 800 nm or; (2) Goat anti-Rbt IR 800nm containing a 1/600 dilution of DRAQ7 and 1/1000 dilution of Sapphire700 for 2-3 hrs at room temperature with shaking in the dark.

Finally, the wells were washed 5 times for 5 min each with 0.1% Tween-20in PBS, with shaking in the dark. The plates were patted dry, thenimaged using the LiCor infrared reader. Odyssey software was used toquantify the pixel intensity within each well.

Example 5 Measurement of Cell Proliferation Inhibition

This example describes an assay that measures the ability of compoundsto inhibit the proliferation of cultured cells. This assay can also beused to assess whether combining two or more compounds producesadditive, synergistic, or antagonistic effects on cell growth. Methodsare according to Examples 1-4 unless otherwise specified.

On day 1, the cells were seeded at 10³ cells per well in 100 μl ofcomplete media under standard conditions.

On day 2, the cells were washed and 100 μl serum free media was added.The cells were then incubated for at least 2 hours before the additionof compound(s). 20 μl of compound(s) was added to the respective wellsand incubated at 37° C., 5% CO₂ for 72 hrs.

On day 5, 50 μl of the MTT dye solution was added to each well. Theplates were incubated at 37° C., 5% CO₂ for 1 hr. The media was thenaspirated and the cells were resuspended in 1000 of DMSO. The plateswere allowed to incubate for 5 min at room temperature with gentleshaking.

The absorbance in each well was determined at 560 nm using a Polarstarplate reader.

Example 6 Measurement of p53 Cellular Activity

This example describes an assay that measures the ability of compoundsto stimulate p53-induced reporter gene activity. In order for p53activity to be observed, the interaction of p53 with MDM2 must beinhibited. Methods are according to Examples 1-5 unless otherwisespecified.

HEK293 cells were transiently transfected with the induciblep53-responsive firefly luciferase reporter and constitutively-expressingRenilla construct. After 16 hrs of transfection at 37° C., 5% CO₂, thetransfection media was removed and 200 μl/well of assay media was added.The cells were then incubated for 8 hrs at 37° C., 5% CO₂.

1 ml of compound(s) was added to the cells and incubated for 16 hrs at37° C. and 5% CO₂. The cells were carefully washed with 200 μl of PBS.20 μl_of lysis buffer was then added to each well and incubated at roomtemperature for 15 minutes with shaking.

The lysis buffer supernatant was transferred to a white/opaque CoStar96-well plate and 100 μl of firefly luceriferase assay substrate wasadded. The luminescence in each well was determined using a Polarstarplate reader.

100 μl of Stop and Glow reagent was added to each well, mixed, and theluminescence generated by the Renilla luciferase was determined using aPolarstar plate reader. The Renilla signal was used to normalize thetransfection efficiency and cell viability.

Example 7 Caspase Activity Measurement of Apoptosis

This example describes an assay that measures the ability of compoundsto increase the activity of caspase 3/7 activity. Methods are accordingto Examples 1-6 unless otherwise specified.

On day 1, 2000 cells were plated per well at 25 μl/well. The cells wereincubated overnight @ 37° C., 5% CO₂.

On day 2, the cells were washed with PBS and 100 μl serum free mediawere added to the cells and incubated overnight.

On day 3, the cells were treated with the compound(s) diluted in250/well of DMEM, with 1 mg/ml BSA. (no FBS). The plates were incubatedfor 5.5 hr at 37° C. The plates were removed from the incubator andallowed to equilibrate to room temperature for 30 min. 25 μl of CapsaseGlo reagent was added to each well. The plates were covered and allowedto incubate at room temperature for 60 min. The luminescence in eachwell was determined using a plate reader.

Example 8 AD4 Compounds Inhibit Cell Proliferation and DemonstrateSynergistic Effect with Tarceva

The following example provides preliminary assessment of the ability ofselected AD4 compounds to inhibit EGF-mediated cell proliferation usingthe A431 cell proliferation assay and to demonstrated synergisticeffects with Tarceva. Methods are according to Examples 1-7 unlessotherwise specified. Compound structures are as disclosed in U.S.application Ser. No. 12/986,146 and WO 2011/085126.

The A431 cell line over-expresses the EGF receptor, and was utilized toassess the ability of AD4 compounds to inhibit EGF-mediated cellproliferation. A number of compounds inhibit cell proliferation in theA431 cell line, with IC₅₀ values ranging from 1.0-3.7 μM (see e.g.,TABLE 6A). Tarceva inhibits cell proliferation with an IC₅₀ value of 0.8μM (see e.g., TABLE 6A).

These results indicate that the AD4 compounds inhibit cell proliferationby interfering with a pathway that can potentiate the effects of the EGFinhibitor.

Furthermore, AD4 compounds demonstrated synergistic effects with Tarceva(see e.g., TABLE 6B). Cl values <0.8 indicate a synergistic effect.Except for AD4-11511, all of the tested AD4 compounds produced synergywhen combined with Tarceva. AD4-1505 and AD4-10963 produced the greatesteffects.

These results indicate that the combination AD4/Tarceva compoundsinhibit cell proliferation by interfering with a pathway that canpotentiate the effects of the EGF receptor inhibitor.

TABLE 6 AD4 Compounds Inhibit Cell Proliferation and DemonstrateSynergistic Effect with Tarceva (A) (B) A431 Cell Proliferation A431Cell Proliferation Compound (IC₅₀, μM) CI Value Tarceva 0.8 AD4-1505 3.70.45 AD4-10963 3.4 0.43 AD4-11511 1.0 0.89 AD4-10482 3.6 0.54 AD4-104831.2 0.70 AD4-10942 3.2 0.60 AD4-10944 1.8 0.52 AD4-10628 1.0 0.76

Example 9 Further Studies Showing AD4 Compounds Inhibit CellProliferation and Demonstrate Synergistic Effect with Tarceva

Based on preliminary results in Example 8, further testing wasconducted. The following example describes the investigation of acompound's ability to inhibit cell proliferation in A431 cells, whichoverexpress EGF receptors. Methods are according to Examples 1-7 unlessotherwise specified. Compound structures are as disclosed in U.S.application Ser. No. 12/986,146 and WO 2011/085126.

The results demonstrate that all AD4 compounds inhibit cellproliferation with an IC₅₀ value <10 μM, and six of the AD4 compoundsinhibit cell proliferation with an IC₅₀ value <1 μM (see e.g., TABLE7A). For comparison, Tarceva, an EGF receptor kinase inhibitor, producedan IC₅₀ value of 0.8 μM. The IC₅₀ values for AD4 compounds compared toTarceva are on the same order of magnitude, displaying similar cellproliferation inhibition.

Furthermore, AD4 compounds in combination with Tarceva were shown toproduce a synergistic effect. A number of compounds in the AD4 serieswere evaluated for their ability to produce synergistic effects (Clvalue <0.8) with Tarceva in the A431 cell proliferation assay (seeExample 5).

Results show that most AD4 compounds tested in combination with Tarcevaproduce a Cl value <0.8, which indicates synergy (see e.g., TABLE 7B).

These results indicate that the AD4 compounds inhibit cell proliferationby interfering with a pathway that can potentiate the effects of EGFreceptor inhibitors.

TABLE 7 A431 Cell Proliferation (A) (B) A431 Cell A431 Cell (C)Proliferation Proliferation p53/MDM2 Compound (IC₅₀Value, μM) CI ValueIC₅₀ Value (μM) AD4-1505 3.7 0.29 3.6 AD4-10315 3.5 0.79 1.5 AD4-103810.62 0.62 AD4-10460 1.1 0.65 AD4-10482 3.6 0.89 18 AD4-10483 1.2 0.754.6 AD4-10484 1.3 0.97 7.4 AD4-10628 1.0 0.57 4.0 AD4-10942 3.2 0.57 18AD4-10944 1.8 0.79 17 AD4-10945 2.7 0.65 4.6 AD4-10963 3.4 0.63 2.0AD4-11511 1.0 0.89 4.3 AD4-12632 6.0 0.29 AD4-12902 1.6 0.7 9.4AD4-12903 1.8 0.67 18 AD4-12905 1.6 0.67 14 AD4-12906 2.8 0.66 4.4AD4-12907 2.6 0.71 9.2 AD4-12908 4.2 0.64 AD4-12909 6.7 0.54 4.2AD4-12910 1.8 0.69 7.5 AD4-12911 1.4 0.76 AD4-12912 1.2 0.70 AD4-129141.4 0.74 AD4-12915 1.1 0.68 AD4-12917 1.6 0.54 3.6 AD4-12918 2.2 0.655.2 AD4-13023 1.8 0.74 AD4-13028 1.8 0.76 AD4-13029 1.8 0.83 AD4-130302.0 0.83 AD4-13031 1.6 0.74 AD4-13032 1.8 0.69 AD4-13033 1.9 0.80AD4-13034 1.9 0.70 AD4-13041 2.0 0.69 AD4-13042 2.2 0.81 AD4-13043 2.10.81 AD4-13049 1.8 0.84 AD4-13051 1.6 0.72 AD4-13052 1.0 0.75 AD4-130531.4 0.78 12 AD4-13054 2.1 0.61 9.0 AD4-13055 1.5 0.75 AD4-13056 1.9 0.63AD4-13057 1.1 0.83 AD4-13058 2.3 0.50 41 AD4-13059 1.0 0.71 AD4-130601.5 0.76 AD4-13061 2.2 0.79 AD4-13062 2.5 0.57 44 AD4-13063 4.4 0.77AD4-13064 5.1 0.64 AD4-13065 4.6 0.61 AD4-13066 5.4 0.60 AD4-13067 1.80.63 8.0 AD4-13068 5.8 0.68 AD4-13069 0.92 0.91 AD4-13070 1.9 0.65 8.0AD4-13071 3.5 0.70 AD4-13072 0.77 0.74 AD4-13073 1.6 0.85 AD4-13075 1.40.83 AD4-13076 1.4 0.71 AD4-13079 1.8 0.91 AD4-13080 2.5 0.68 6.0AD4-13082 1.9 0.90 AD4-13085 1.7 0.86 AD4-13086 1.5 0.80 AD4-13087 1.80.70 AD4-13088 1.5 0.76 AD4-13089 1.9 0.67 16 AD4-13091 1.6 0.89AD4-13092 1.5 0.79 AD4-13093 1.8 0.80 AD4-13094 1.3 0.76 AD4-13095 0.830.75 AD4-13096 3.0 0.61 AD4-13098 1.6 0.86 AD4-13099 2.1 0.61 10AD4-13101 2.1 0.66 20 AD4-13102 1.4 0.75 AD4-13103 1.4 0.79 AD4-131042.8 0.67 AD4-13106 0.65 0.75 AD4-13107 1.7 0.64 AD4-13108 1.6 0.84AD4-13109 1.5 0.88 AD4-13111 0.74 0.95 AD4-13112 2.1 0.73 AD4-13113 1.60.59 9.0 AD4-13114 0.86 0.81 AD4-13115 1.6 0.74 AD4-13116 1.8 0.52 13AD4-13117 1.3 0.72 AD4-13118 1.5 0.60 24 AD4-13121 1.9 0.75 AD4-131221.6 0.84 AD4-13123 1.3 0.71 AD4-13124 1.6 0.76 AD4-13125 1.7 0.70AD4-13132 1.9 0.82 AD4-13139 1.4 0.75 AD4-13145 2.4 0.86 AD4-13177 2.00.9 AD4-13181 2.2 0.89 AD4-13193 4.7 0.92 24 AD4-13194 5.3 0.97 26AD4-13195 5.8 0.81 25 AD4-13196 5.0 0.69 10 AD4-13197 3.4 0.63 18AD4-13198 3.0 0.87 AD4-13199 2.0 1.1 AD4-13200 1.5 0.97 AD4-13201 2.00.82 AD4-13202 1.8 0.87 10 AD4-13243 2.2 0.56 8.5

Example 10 AD4 Compounds Inhibit Binding of p53 and MDM2

The following example demonstrates the ability of AD4 compounds toinhibit the binding of p53 and MDM2 in a biochemical assay. Methods areaccording to Examples 1-7 unless otherwise specified. Compoundstructures are as disclosed in U.S. application Ser. No. 12/986,146 andWO 2011/085126.

The results, which are summarized in TABLE 9, indicate that many of theAD4 compounds inhibit the binding of p53 and MDM2 with an IC₅₀ value <10μM.

TABLE 9 Inhibition of p53/MDM2 Binding Compound IC₅₀ Value (μM) AD4-15054.2 AD4-1969 26 AD4-1973 4.6 AD4-1976 5.6 AD4-1978 16 AD4-1991 5.0AD4-1997 8.0 AD4-10013 16 AD4-10016 5.5 AD4-10017 3.5 AD4-10028 7.1AD4-10031 6.3 AD4-10037 7.6 AD4-10051 19 AD4-10052 14 AD4-10053 40AD4-10055 19 AD4-10068 18 AD4-10086 5.7 AD4-10087 7.7 AD4-10101 7.0AD4-10108 10 AD4-10143 21 AD4-10144 5.8 AD4-10315 1.5 AD4-10427 21AD4-10430 5.0 AD4-10460 6.2 AD4-10466 13 AD4-10482 18 AD4-10483 4.6AD4-10484 7.4 AD4-10487 21 AD4-10546 3.1 AD4-10547 4.3 AD4-10550 9.3AD4-10551 10 AD4-10602 18 AD4-10628 4.0 AD4-10936 16 AD4-10938 55AD4-10939 8.6 AD4-10942 18 AD4-10944 17 AD4-10945 4.6 AD4-10952 0.33AD4-10955 20 AD4-10957 61 AD4-10958 10 AD4-10959 3.4 AD4-10960 17AD4-10961 0.92 AD4-10963 2.0 AD4-10968 5.4 AD4-10974 25 AD4-11000 17AD4-11017 3.4 AD4-11042 18 AD4-11057 20 AD4-11072 49 AD4-11073 20AD4-11102 17 AD4-11103 4.2 AD4-11105 21 AD4-11151 4.3 AD4-11153 3.4AD4-12902 9.4 AD4-12903 18 AD4-12905 14 AD4-12906 4.4 AD4-12907 9.2AD4-12909 4.2 AD4-12910 7.5 AD4-12917 3.6 AD4-12918 5.2 AD4-12941 9.5AD4-13053 12 AD4-13054 9.0 AD4-13058 41 AD4-13062 44 AD4-13067 8.0AD4-13070 8.0 AD4-13080 6.0 AD4-13089 16 AD4-13099 10 AD4-13101 20AD4-13113 9.0 AD4-13116 13 AD4-13118 24 AD4-13193 24 AD4-13194 26AD4-13195 26 AD4-13196 10 AD4-13197 18 AD4-13202 10 AD4-13208 13AD4-13210 20 AD4-13214 20 AD4-13219 30 AD4-13243 8.5 AD4-13256 9.7AD4-13262 5.0 AD4-13263 4.7 AD4-13264 4.5 AD4-13265 3.4

Example 11 Combination AD4/Tarceva Inhibit Binding of p53 and MDM2

The following example demonstrates that combination AD4/Tarcevacompounds that inhibit cell proliferation with synergistic effects alsoinhibit p53/MDM2 binding. Methods are according to Examples 1-7 unlessotherwise specified. Compound structures are as disclosed in U.S.application Ser. No. 12/986,146 and WO 2011/085126.

The p53/MDM2 results for the compounds (equivalent compounds alsoevaluated in the A431 cell proliferation assay) are shown in TABLE 6C.These results show that compounds that inhibit cell proliferation in acell line that over-expresses the EGF receptor, and that produce synergywith the EGF receptor inhibitor, Tarceva, are able to inhibit thebinding of p53 and MDM2. As a result, compounds that inhibit the bindingof p53 and MDM2 may provide a novel therapeutic approach for enhancingthe activity of compounds that inhibit EGF receptor activity, such asTarceva.

Example 12 AD4 Compounds Inhibit Binding of p53 with MDM2

The following example demonstrates a series of identified compounds thatinhibit binding of p53 with MDM2. Methods are according to Examples 1-7unless otherwise specified. The assay was performed as described inExample 1, except that recombinant full length human N-terminal GST-MDM2was used and obtained from ABNOVA, and the wild type, full length humanN-terminal 6-his p53 was purchased from Fisher Scientific Co. Compoundstructures are as disclosed in U.S. application Ser. No. 12/986,146 andWO 2011/085126.

The compounds studied in this example were AD4 1505, AD4 10963, AD411511, AD4 10482, AD4 10942, AD4 10944, and AD4 10628.

The most potent of the compounds studied in this example (see e.g.FIG. 1) were AD4-10963 and AD4-1505, which inhibit binding of theproteins with EC₅₀ values of 100 and 270 nM, respectively.

Example 13 AD4 Compounds Inhibit Interaction of p53 and MDM2

The following example demonstrates selected compounds that stimulatedp53 reporter gene activity using the p53 reporter gene assay. Methodsare according to Examples 1-7 unless otherwise specified. Compoundstructures are as disclosed in U.S. application Ser. No. 12/986,146 andWO 2011/085126.

Assessed compounds were chosen from those that potently inhibit activityin the p53/MDM2 biochemical assay.

At a concentration of 10 μM, AD4-1505, AD4-10953 and AD4-10944stimulated p53 reporter gene activity by 42.5-, 9.7- and 7.5-fold,respectively. These results show that the compounds effectively inhibitthe interaction of p53 and MDM2 in the cell.

Example 14 Correlation of p53/MDM2 Inhibition and Ability of AD4Compounds to Produce Synergy with Tarceva

A correlation plot shows the relationship between the ability ofcompounds to inhibit p53/MDM2 binding (see Example 13) and the abilityto produce synergy with Tarceva (see Example 11) in the A431 cellproliferation assay. Compound structures are as disclosed in U.S.application Ser. No. 12/986,146 and WO 2011/085126.

Methods are according to Examples 1-7 unless otherwise specified.

A significant correlation was observed, with an Revalue=0.70 (p<0.05)(see e.g., FIG. 3).

Example 15 AD4 Compounds Induce Apoptosis as Measured by DNAFragmentation

The following example demonstrates the ability of compounds to induceapoptosis evaluated by measuring the ability of compounds to induce DNAfragmentation. Methods are according to Examples 1-7 unless otherwisespecified. Compound structures are as disclosed in U.S. application Ser.No. 12/986,146 and WO 2011/085126.

The ability of compounds to induce DNA fragmentation was evaluated as anadditional measure of apoptosis in A549 lung cancer cells. The percentincrease in DNA fragmentation produced by compounds at a concentrationof 1 and 10 μM were compared to background, and to the response producedby 500 nM staurosporin.

The results for those compounds that produced a 20% increase in responserelative to staurosporin are summarized in TABLE 10. A total of 28compounds were found active.

TABLE 10 Induction of DNA Fragmentation in A549 Cells % Inc. Relative %Inc. over to Staurosporin Background (500 nM) Compound 1 μM 10 μM 1 μM10 μM AD4-13123 366 476 40 51 AD4-13130 565 614 58 65 AD4-13134 573 46956 46 AD4-13147 406 462 48 52 AD4-13161 254 688 25 68 AD4-13164 390 57938 55 AD4-13165 172 582 24 74 AD4-13172 355 782 32 70 AD4-13178 395 58147 68 AD4-13185 285 576 32 62 AD4-13187 388 543 47 62 AD4-13224 296 66225 59 AD4-13225 508 458 48 42 AD4-13229 531 667 68 84 AD4-13243 551 71670 91 AD4-13260 148 310 26 52 AD4-13261 184 538 20 63 AD4-13277 484 96250 103 AD4-13290 172 637 22 79 AD4-13292 200 854 20 86 AD4-13299 592 68854 62 AD4-13303 403 828 35 69 AD4-13309 444 858 38 71 AD4-13311 506 87742 72 AD4-13314 803 735 66 62 AD4-13316 486 902 40 75 AD4-13317 757 89257 67 AD4-13323 621 661 57 60

Example 16 AD4 Compounds Induce Apoptosis as Measured by Increases DNAFragmentation

The following example demonstrates the activity of compounds on p53using a p53 In Cell Western assay. Methods are according to Examples 1-7unless otherwise specified. Compound structures are as disclosed in U.S.application Ser. No. 12/986,146 and WO 2011/085126.

Using antibodies to total p53 or p53 phosphorylated at S15, the abilityof compounds to alter the expression of total p53 in the cell, or theamount of phosphorylated S15 p53, were assessed.

The results indicate that AD4-13243, which increased DNA fragmentationand increased phosphorylated p53 in the proteome profile assay,significantly increased both total p53 expression and S15p-p53 atconcentrations of about 1-3 μM. These results demonstrate that AD4-13243can induce apoptosis by increasing the expression of p53.

Example 17 Combination AD4/Tarceva Synergistically Induce Apoptosis

The following example demonstrates the ability of compounds to induceapoptosis evaluated by the measurement of the ability of compounds toinduce caspase 3/7 activity. Methods are according to Examples 1-7unless otherwise specified. Compound structures are as disclosed in U.S.application Ser. No. 12/986,146 and WO 2011/085126.

In the caspase assay, which was conducted in A431 cells, compounds wereevaluated for their ability to induce apoptosis, relative to the effectproduced by 8 μM Tarceva alone. Additionally, cells were evaluated forsynergistic effects of AD4 compounds in combination with Tarceva. Themaximal effect (100%) was defined using 500 nM staurosporin (a compoundcommonly used to induce apoptosis, in vitro). Synergy was determined bydetermining whether the effect of the combination of Tarceva plus theAD4 compound produced a greater effect than the sum of the effect ofTarceva alone and the AD4 compound alone.

The results of these studies, which are summarized in TABLE 11, indicatethat several compounds induce apoptosis as measured by an increase incaspase. Furthermore, these compounds produce synergy with Tarceva inthis assay.

TABLE 11 Caspase 3/7 Activity % Inc. Relative to Synergy with CompoundTarceva Tarceva AD4-13072 52% Yes AD4-13095 27% Yes AD4-13107 48% YesAD4-13181 73% Yes AD4-13185 37% Yes AD4-13192 131%  Yes AD4-13240 221% Yes AD4-13254 67% Yes

Example 18 Combination AD4/Tarceva Compounds Induce Apoptosis with noMeasurable Cytotoxicity

The following example demonstrates selected compounds that induceapoptosis. Methods are according to Examples 1-7 unless otherwisespecified. Compound structures are as disclosed in U.S. application Ser.No. 12/986,146 and WO 2011/085126.

Compounds that inhibit proliferation of cancer cells produce acytostatic effect, whereas compounds that induce apoptosis, or celldeath, of cancer cells are cytotoxic.

Compounds were evaluated for induced apoptosis measured by the inductionof caspase 3/7 activity (see Example 7). The ability of compounds toinduce caspase 3/7 activity in A431 cells at a concentration of 10 μM iscompared to the effect produced by 10 μM Tarceva.

The results (see e.g., TABLE 12) indicate that all of the compoundsinduce apoptosis, some of which (e.g. AD4-10628, AD4-10483 andAD4-11511) produce an effect equal to or greater than Tarceva. None ofthese compounds were found to produce measurable cytotoxicity in A431cells.

TABLE 12 % Increase Relative to Compound Tarceva AD4-1505 45% AD4-1096338% AD4-11511 137%  AD4-10482 21% AD4-10483 112%  AD4-10942 72%AD4-10944 72% AD4-10628 145% 

The effect of the compounds when combined with Tarceva in the apoptosisassay was also evaluated (see e.g., FIG. 4). The combination ofAD4-10483 and Tarceva demonstrated synergistic effects.

The data in this example shows that for all AD4 and Tarceva compoundsstudied, all induced apoptosis as individual compounds, howeversurprising results of synergy was observed when in combination.

Example 19 AD4 Increased Phosphorylated p53 Levels Measured by ProteomeProfiling

The following example demonstrates the mechanism of action of thecurrent AD4 compound series. Methods are according to Examples 1-7unless otherwise specified. Compound structures are as disclosed in U.S.application Ser. No. 12/986,146 and WO 2011/085126.

First, several compounds were evaluated in a proteome profiling arraysystem containing antibodies to 35 apoptosis-related proteins. In thisassay (see Example 3), the relative expression levels of these proteinsin cell extracts are measured from treated and non-treated A549 cells.

The results from this study indicate that 6 AD4 compounds (AD4-13178,AD4-13225, AD4-13243, AD4-13130, AD4-13229 and AD4-13165) at aconcentration of 5 μM, increased all three phosphorylated forms of p53,similar to nutlin, which inhibits the binding of p53 and MDM2.

Surprisingly, the pattern of expression produced by the AD4 compoundswas different from nutlin (e.g., nutlin increased the expression of BADand Bax, whereas the AD4 compounds did not), demonstrating that the AD4compounds produce their effect on p53 by a different mechanism thannutlin. More importantly, all six of the compounds that increased thephosphorylated forms of p53 in the proteome profiler assay increasedapoptosis, as measured by DNA fragmentation.

AD4 compounds in this example increased all three phosphorylated formsof p53 which inhibit the binding of p53 and MDM2.

What is claimed is:
 1. A composition comprising: (a) an MDM2 inhibitor;and (b) an EGFR inhibitor, wherein the MDM2 inhibitor is a compoundhaving a formula of

or a stereoisomer or pharmaceutically acceptable salt thereof; X isselected from the group consisting of hydrogen, 2-Methyl, 5-Chloro,5-Nitro, and 6-Hydroxyl; R¹ is selected from the group consisting of (i)a 2-Pyridyl ring of Formula (3)

wherein R²³ is selected from the group consisting of hydrogen; fluoro;chloro; trifluoromethyl; methyl; ethyl; and methoxy; R³ is selected fromthe group consisting of hydrogen; fluoro; chloro; methyl; ethyl;methoxy; a straight chain or branched C-1 to C-4 lower alkyl optionallycontaining unsaturation; a C-1 to C-6 cycloalkyl optionally containingunsaturation or one oxygen or nitrogen atom; aryl comprising a phenyl orheteroaryl five or six membered ring containing from 1 to 4 N, O, or Satoms; and alkoxy —OR¹⁰ where R¹⁰ is a straight chain or branched C-1 toC-4 lower alkyl optionally containing unsaturation or a C-1 to C-6cycloalkyl optionally containing unsaturation or one oxygen or nitrogenatom; R²⁴ is selected from the group consisting of hydrogen; fluoro;chloro; and trifluoromethyl; and R⁴ is selected from the groupconsisting of hydrogen; methyl; a straight chain or branched C-1 to C-4lower alkyl optionally containing unsaturation; a C-1 to C-6 cycloalkyloptionally containing unsaturation or one oxygen or nitrogen atom; arylcomprising a phenyl or heteroaryl five or six membered ring containingfrom 1 to 4 N, O, or S atoms; and alkoxy —OR¹⁰ where R¹⁰ is a straightchain or branched C-1 to C-4 lower alkyl optionally containingunsaturation or a C-1 to C-6 cycloalkyl optionally containingunsaturation or one oxygen or nitrogen atom; (ii) a 3-Pyridyl ring ofFormula (4)

wherein R⁵, R⁶, and R⁷ are independently selected from the groupconsisting of hydrogen; trifluoromethyl; a straight chain or branchedC-1 to C-4 lower alkyl optionally containing unsaturation, optionallycontaining one or more halogens; a C-1 to C-6 cycloalkyl optionallycontaining unsaturation or one oxygen or nitrogen atom; Aryl comprisinga phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms; andAlkoxy —OR¹⁰ where R¹⁰ is a straight chain or branched C-1 to C-4 loweralkyl optionally containing unsaturation, optionally containing one ormore halogens; or a C-1 to C-6 cycloalkyl optionally containingunsaturation or one oxygen or nitrogen atom; and (iii) a 4-Pyridyl ringof Formula (5)

wherein R⁸ and R⁹ are independently selected from the group consistingof a straight chain or branched C-1 to C-4 lower alkyl optionallycontaining unsaturation; a C-1 to C-6 cycloalkyl optionally containingunsaturation or one oxygen or nitrogen atom; aryl comprising a phenyl orheteroaryl containing from 1 to 4 N, O, or S atoms; and alkoxy —OR¹⁰where R¹⁰ is a straight chain or branched C-1 to C-4 lower alkyloptionally containing unsaturation or a C-1 to C-6 cycloalkyl optionallycontaining unsaturation or one oxygen or nitrogen atom; (iv) a phenylring substituted with one or more groups selected from a straight chainor branched C-1 to C-4 lower alkyl optionally containing unsaturation; aC-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygenor nitrogen atom; aryl comprising a phenyl or heteroaryl containing from1 to 4 N, O, or S atoms; alkoxy —OR¹⁰ where R¹⁰ is a straight chain orbranched C-1 to C-4 lower alkyl optionally containing unsaturation or aC-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygenor nitrogen atom; trifluoromethyl; trifluoromethoxy; difluoromethoxy;3,4-methylenedioxy; 2,3-methylenedioxy; nitro; and halogen; and (v) anunsubstituted heteroaryl five or six membered ring containing from 1 to4 N, O, or S atoms; (vi) a substituted heteroaryl five or six memberedring containing from 1 to 4 N, O, or S atoms substituted with one ormore groups selected from the group consisting of straight chain orbranched C-1 to C-4 lower alkyl optionally containing unsaturation; C-1to C-6 cycloalkyl optionally containing unsaturation or one oxygen ornitrogen atom; aryl comprising a phenyl or heteroaryl five or sixmembered ring containing from 1 to 4 N, O, or S atoms; and alkoxy —OR¹⁰where R¹⁰ is a straight chain or branched C-1 to C-4 lower alkyloptionally containing unsaturation or a C-1 to C-6 cycloalkyl optionallycontaining unsaturation or one oxygen or nitrogen atom; and R² isselected from the group consisting of (i) an unsubstituted phenyl ringor a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position withone or more groups independently selected from the group consisting ofstraight chain or branched C-1 to C-4 lower alkyl optionally containingunsaturation; C-1 to C-6 cycloalkyl optionally containing unsaturationor one oxygen or nitrogen atom; aryl comprising a phenyl or heteroarylfive or six membered ring containing from 1 to 4 N, O, or S atoms;hydroxy; alkoxy —OR¹⁰ where R¹⁰ is a straight chain or branched C-1 toC-4 lower alkyl optionally containing unsaturation or a C-1 to C-6cycloalkyl optionally containing unsaturation or one oxygen or nitrogenatom; 2,3-methylenedioxy; 3,4-methylenedioxy; trifluoroethoxy;dialkylamino having formula —NR₁₃R₁₄ wherein R₁₃ and R₁₄ areindependently selected from hydrogen; straight chain or branched C-1 toC-4 lower alkyl optionally containing unsaturation; trifluoromethyl;trifluoromethoxy; difluoromethoxy; 3,4-methylenedioxy;2,3-methylenedioxy; nitro; and halogen; (ii) a 2-thiophene ring ofFormula (8) wherein R¹⁵, R¹⁶, and R¹⁷ are independently selected fromthe group consisting of hydrogen; straight chain or branched C-1 to C-4lower alkyl optionally containing unsaturation; C-1 to C-6 cycloalkyloptionally containing unsaturation or one oxygen or nitrogen atom;alkoxy —OR¹⁰ where R¹⁰ is a straight chain or branched C-1 to C-4 loweralkyl optionally containing unsaturation or a C-1 to C-6 cycloalkyloptionally containing unsaturation or one oxygen or nitrogen atom;dialkylamino; trifluoromethyl; difluoromethyl; trifluoromethoxy; andhalogen

(iii) a 3-thiophene ring of Formula (9) wherein R¹⁸, R¹⁹, and R²⁰ areindependently selected from the group consisting of straight chain orbranched C-1 to C-4 lower alkyl optionally containing unsaturation; C-1to C-6 cycloalkyl optionally containing unsaturation or one oxygen ornitrogen atom; alkoxy —OR¹⁰ where R¹⁰ is a straight chain or branchedC-1 to C-4 lower alkyl optionally containing unsaturation or a C-1 toC-6 cycloalkyl optionally containing unsaturation or one oxygen ornitrogen atom; dialkylamino; trifluoromethyl; difluoromethyl;trifluoromethoxy; and halogen

(iv) an unsubstituted 2-Pyridyl ring or a 2-Pyridyl ring substituted at4- or 6-position of the pyridine ring with one or more groupsindependently selected from the group consisting of straight chain orbranched C-1 to C-4 lower alkyl optionally containing unsaturation andC-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygenor nitrogen atom; (v) an unsubstituted 3-Pyridyl ring or a 3-Pyridylring substituted at the 2-, 4- or 6-position of the pyridine ring withone or more groups independently selected from the group consisting ofstraight chain or branched C-1 to C-4 lower alkyl optionally containingunsaturation and C-1 to C-6 cycloalkyl optionally containingunsaturation or one oxygen or nitrogen atom; and (vi) an unsubstituted4-Pyridyl ring or a 4-Pyridyl ring substituted at the 2- or 6-positionof the pyridine ring with one or more groups independently selected fromthe group consisting of straight chain or branched C-1 to C-4 loweralkyl optionally containing unsaturation and C-1 to C-6 cycloalkyloptionally containing unsaturation or one oxygen or nitrogen atom; or astereoisomer or pharmaceutically acceptable salt thereof.
 2. Thecomposition of claim 1, wherein a combination of the MDM2 inhibitor andthe EGFR inhibitor results in a synergistic reduction in cellproliferation in a tumor of the subject or a synergistic increase inapoptosis in a tumor of the subject as compared to administration ofeither the MDM2 inhibitor or the EGFR inhibitor alone.
 3. Thecomposition of claim 1, wherein the EGFR inhibitor is selected from thegroup consisting of cetuximab, panitumumab, nimotuzumab, zalutumumab,matuzumab, potato carboxypeptidase inhibitor, gefitinib, lapatinib, anderlotinib, or a combination thereof.
 4. The composition of claim 1,wherein the EGFR inhibitor is erlotinib.
 5. The composition of claim 1,wherein the MDM2 inhibitor (i) inhibits MDM2 activity; (ii) increasesphosphorylated p53; (iii) re-activates p53; or (iv) inhibits binding ofp53 and MDM2; or a combination thereof.
 6. The composition of claim 1,wherein the MDM2 inhibitor is a compound having a formula of:

or a stereoisomer or pharmaceutically acceptable salt thereof; wherein,X is selected from the group consisting of hydrogen, 2-Methyl, and5-Chloro, R¹ is selected from the group consisting of (i) a 2-Pyridylring of Formula (3)

wherein R²³ is selected from the group consisting of hydrogen; fluoro;chloro; and methyl; R³ is selected from the group consisting ofhydrogen; chloro; and methyl; R²⁴ is selected from the group consistingof hydrogen; fluoro; chloro; and trifluoromethyl; and R⁴ is selectedfrom the group consisting of hydrogen; and methyl; (ii) a 3-Pyridyl ringof Formula (4)

wherein R⁵, R⁶, and R⁷ are independently selected from the groupconsisting of hydrogen and trifluoromethyl and R² is selected from thegroup consisting of (i) an unsubstituted phenyl ring or a phenyl ringsubstituted at the 2-, 3-, 4-, 5- or 6-position with one or more groupsindependently selected from the group consisting of methyl; methoxy;ethoxy; trifluoroethoxy; trifluoromethyl; hydroxy; trifluoromethoxy; Cl;and F; (ii) a 2-thiophene ring of Formula (8) wherein R¹⁵, R¹⁶, and R¹⁷are independently selected from the group consisting of hydrogen andmethyl

or a stereoisomer or pharmaceutically acceptable salt thereof.
 7. Thecomposition of claim 1, wherein the MDM2 inhibitor comprises a compoundselected from the group consisting of:


8. A kit comprising (a) a first composition comprising an MDM2 inhibitorand an EGFR inhibitor or (b) a second composition comprising an MDM2inhibitor and a third composition comprising an EGFR inhibitor accordingto definitions of claim 1.